Assays and Related Methods, Kits, and Devices

Abstract

The present disclosure relates to correlating the ratio of determined levels of fetuin A fragment to determined levels of urinary creatinine with the likelihood of graft failure in the recipient.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 441,751, entitled "ASSAY AND RELATED METHODS, KITS AND DEVICES," filed January 27, 2023, and U.S. Patent Application No. 63 / 532,655, entitled "ASSAY AND RELATED METHODS, KITS AND DEVICES," filed August 14, 2023, each of which is incorporated herein by reference in its entirety. [Background technology]

[0002] End-stage kidney disease (ESKD) is a leading cause of morbidity and mortality and a major global health burden. Kidney transplantation remains the most preferred treatment for ESKD to date because it offers a better quality of life and is cost-effective compared to other kidney replacement therapies. Summary of the Invention

[0003] This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. It is not intended to identify key or essential aspects of the claimed subject matter.

[0004] All features of exemplary embodiments described in this disclosure that are not mutually exclusive may be combined with one another. Elements of one embodiment may be utilized in other embodiments without further recitation. Other aspects and features of the present invention will become apparent to those skilled in the art upon review of the following description of specific embodiments in conjunction with any accompanying drawings.

[0005] The disclosure may also relate to a method of preparing an assay, the method comprising providing a first solution comprising a urine sample from a kidney transplant recipient after kidney transplantation and a first reagent for interacting with post-translationally modified fetuin in the urine fragment to determine a level of fetuin A fragment in the urine sample, providing a second solution comprising the urine sample and a second reagent for interacting with urinary creatinine to determine a level of urinary creatinine in the urine sample, and determining a level of urinary creatinine in response to the determined level of fetuin A fragment and the determined level of urinary creatinine. and correlating the ratio of the determined level of fetuin-A fragment to the level with a likelihood of graft failure in the recipient, wherein a ratio greater than about 20 ng / mg, greater than about 21 ng / mg, greater than about 22 ng / mg, greater than about 23 ng / mg, more preferably greater than about 23.5 ng / mg, and greater than about 20 ng / mg indicates a higher likelihood of graft failure over a period of about 10 years or less, and wherein the first solution and the second solution can be the same solution or different solutions from each other.

[0006] In some embodiments, a ratio greater than about 20 ng / mg, greater than about 21 ng / mg, greater than about 22 ng / mg, greater than about 23 ng / mg, more preferably greater than about 23.5 ng / mg, and more preferably greater than about 24 ng / mg, triggers medical intervention to forestall graft failure.

[0007] In some embodiments, the higher likelihood may be greater than a lower likelihood of graft decline over a period of about 10 years or less in a second subject having a lower ratio of determined level of fetuin-A fragment to level of urinary creatinine of less than about 16 ng / mg, less than about 15.5 ng / mg, less than about 15 ng / mg, less than about 14.5 ng / mg, or less than about 14 ng / mg.

[0008] In some embodiments, graft deterioration may be indicated by a doubling of serum creatinine, the need for kidney retransplantation, or the need for dialysis.

[0009] In some embodiments, reduced graft function may be indicated by a decrease in estimated glomerular filtration rate (eGFR).

[0010] In some embodiments, interacting with a fetuin A fragment may include binding to a fetuin A fragment.

[0011] In some embodiments, interacting with urinary creatinine may include binding to urinary creatinine.

[0012] In some embodiments, at least one of the first and second reagents may comprise an antibody.

[0013] In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 25 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 25.5 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 26 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 27 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 27.5 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 28 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 28.5 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 28.7 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 28.8 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 28.9 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 29.0 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 29.1 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 29.2 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 29.3 ng / mg.

[0014] In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 30 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 34 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 35 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 37 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 38 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 40 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 50 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 60 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 65 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 70 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 71 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 72 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 73 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 75 ng / mg.

[0015] In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 80 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 85 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 90 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 91 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 92 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 93 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 94 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 90 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 95 ng / mg.

[0016] In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 13 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 12 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 11 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 10 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 9 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 8.8 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 8.5 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 8.4 ng / mg. In some embodiments, the second ratio less than about 14 ng / mg can be less than about 8.2 ng / mg.

[0017] In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 8 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 7.5 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 7.4 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 3.3 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 7.3 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 7.2 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 7.1 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 7 ng / mg. In some embodiments, the second ratio less than about 14 ng / mg can be less than about 6 ng / mg.

[0018] In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 5 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 4 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 3.9 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 3.8 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 3.7 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 3.6 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 3.5 ng / mg.

[0019] In some embodiments, the higher likelihood of graft failure may be about 5% or greater. In some embodiments, the higher likelihood of graft failure may be about 8% or greater. In some embodiments, the higher likelihood of graft failure may be about 10% or greater. In some embodiments, the higher likelihood of graft failure may be about 15% or greater. In some embodiments, the higher likelihood of graft failure may be about 16% or greater.

[0020] In some embodiments, the higher likelihood of graft failure may be about 20% or greater. In some embodiments, the higher likelihood of graft failure may be about 22% or greater. In some embodiments, the higher likelihood of graft failure may be about 25% or greater. In some embodiments, the higher likelihood of graft failure may be about 30% or greater. In some embodiments, the higher likelihood of graft failure may be about 32% or greater. In some embodiments, the higher likelihood of graft failure may be about 35% or greater. In some embodiments, the higher likelihood of graft failure may be about 36% or greater. In some embodiments, the higher likelihood of graft failure may be about 40% or greater. In some embodiments, the higher likelihood of graft failure may be about 45% or greater. In some embodiments, the higher likelihood of graft failure may be about 50% or greater.

[0021] In some embodiments, the higher likelihood of graft dysfunction may be about 5% to about 50%. In some embodiments, the higher likelihood of graft dysfunction may be about 10% to about 45%. In some embodiments, the higher likelihood of graft dysfunction may be about 20% to about 40%. In some embodiments, the higher likelihood of graft dysfunction may be about 25% to about 35%. In some embodiments, the higher likelihood of graft dysfunction may be about 25% to about 30%. In some embodiments, the higher likelihood of graft dysfunction may be about 10% to about 20%. In some embodiments, the higher likelihood of graft dysfunction may be about 15% to about 25%. In some embodiments, the higher likelihood of graft dysfunction may be about 20% to about 30%. In some embodiments, the higher likelihood of graft dysfunction may be about 30% to about 40%. In some embodiments, the higher likelihood of graft dysfunction may be about 35% to about 45%. In some embodiments, the higher likelihood of graft failure may be about 40% to about 50%.

[0022] In some embodiments, graft failure occurs after 2 years. In some embodiments, graft failure occurs after 3 years. In some embodiments, graft failure occurs after 4 years. In some embodiments, graft failure occurs after 5 years. In some embodiments, graft failure occurs after 6 years. In some embodiments, graft failure occurs after 7 years. In some embodiments, graft failure occurs after 8 years. In some embodiments, graft failure occurs after 9 years. In some embodiments, graft failure occurs within 7 years. In some embodiments, graft failure occurs within 8 years. In some embodiments, graft failure occurs within 9 years.

[0023] In some embodiments, the method may further include assessing the probability of graft failure based on the ratio and at least one other marker.

[0024] In some embodiments, the at least one other marker may include age, sex, time since transplant at enrollment, urinary albumin to creatine ratio (UACR), estimated glomerular filtration rate (eGFR) based on the creatinine and cystatin C-based CKD-EPI equation, presence of human leukocyte antigen, or any combination thereof.

[0025] In some embodiments, the assessed probability may represent at least about 70 percent (%) of the area under the receiver operating characteristic (ROC) curve (AUC). In some embodiments, the assessed probability may represent at least about 80 percent (%) of the AUC. In some embodiments, the assessed probability may represent at least about 85 percent (%) of the AUC. In some embodiments, the assessed probability may represent at least about 90 percent (%) of the AUC. In some embodiments, the assessed probability may represent at least about 95 percent (%) of the AUC.

[0026] The present disclosure may also relate to an assay kit for determining the likelihood of progressive renal function decline, the assay kit comprising: a first solution including a first reagent for interacting with post-translationally modified fetuin-A fragments in urine fragments to indicate a level of fetuin-A fragments in a urine sample from a kidney transplant recipient after kidney transplantation; a second solution including a second reagent for interacting with urinary creatinine to indicate a level of urinary creatinine in the urine sample; and a second solution including a second reagent for determining the level of fetuin-A fragments and the level of urinary creatinine in the urine sample, and a device for determining a ratio of a determined level of fetuin A fragment to a determined level, wherein a ratio greater than about 20 ng / mg, greater than about 21 ng / mg, greater than about 22 ng / mg, greater than about 23 ng / mg, more preferably greater than about 23.5 ng / mg, more preferably greater than about 24 ng / mg, greater than about 20 ng / mg indicates a higher likelihood of graft function deterioration over a period of about 10 years or less, and wherein the first solution and the second solution can be the same solution or different solutions from each other.

[0027] In some embodiments, a ratio greater than about 20 ng / mg, greater than about 21 ng / mg, greater than about 22 ng / mg, greater than about 23 ng / mg, more preferably greater than about 23.5 ng / mg, and more preferably greater than about 24 ng / mg, triggers medical intervention to forestall graft failure.

[0028] In some embodiments, the higher likelihood may be greater than a lower likelihood of graft decline over a period of about 10 years or less in a second subject having a lower ratio of determined level of fetuin-A fragment to level of urinary creatinine of less than about 16 ng / mg, less than about 15.5 ng / mg, less than about 15 ng / mg, less than about 14.5 ng / mg, or less than about 14 ng / mg.

[0029] In some embodiments, graft deterioration may be indicated by a doubling of serum creatinine, the need for kidney retransplantation, or the need for dialysis.

[0030] In some embodiments, reduced graft function may be indicated by a decrease in estimated glomerular filtration rate (eGFR).

[0031] In some embodiments, interacting with a fetuin A fragment may include binding to a fetuin A fragment.

[0032] In some embodiments, interacting with urinary creatinine may include binding to urinary creatinine.

[0033] In some embodiments, at least one of the first and second reagents may comprise an antibody.

[0034] In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 25 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 25.5 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 26 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 27 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 27.5 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 28 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 28.5 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 28.7 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 28.8 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 28.9 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 29.0 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 29.1 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 29.2 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 29.3 ng / mg.

[0035] In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 30 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 34 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 35 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 37 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 38 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 40 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 50 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 60 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 65 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 70 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 71 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 72 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 73 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 75 ng / mg.

[0036] In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 80 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 85 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 90 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 91 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 92 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 93 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 94 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 90 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 95 ng / mg.

[0037] In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 13 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 12 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 11 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 10 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 9 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 8.8 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 8.5 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 8.4 ng / mg. In some embodiments, the second ratio less than about 14 ng / mg can be less than about 8.2 ng / mg.

[0038] In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 8 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 7.5 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 7.4 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 3.3 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 7.3 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 7.2 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 7.1 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 7 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 6 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 5 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 4 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 3.9 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 3.8 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 3.7 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 3.6 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 3.5 ng / mg.

[0039] In some embodiments, the higher likelihood of graft failure may be about 5% or greater. In some embodiments, the higher likelihood of graft failure may be about 8% or greater. In some embodiments, the higher likelihood of graft failure may be about 10% or greater. In some embodiments, the higher likelihood of graft failure may be about 15% or greater. In some embodiments, the higher likelihood of graft failure may be about 16% or greater. In some embodiments, the higher likelihood of graft failure may be about 20% or greater. In some embodiments, the higher likelihood of graft failure may be about 22% or greater. In some embodiments, the higher likelihood of graft failure may be about 25% or greater. In some embodiments, the higher likelihood of graft failure may be about 30% or greater. In some embodiments, the higher likelihood of graft failure may be about 32% or greater. In some embodiments, the higher likelihood of graft failure may be about 35% or greater. In some embodiments, the higher likelihood of graft failure may be about 36% or greater. In some embodiments, the higher likelihood of graft failure may be about 40% or greater. In some embodiments, the higher likelihood of graft failure may be about 45% or greater, hi some embodiments, the higher likelihood of graft failure may be about 50% or greater.

[0040] In some embodiments, the higher likelihood of graft dysfunction may be about 5% to about 50%. In some embodiments, the higher likelihood of graft dysfunction may be about 10% to about 45%. In some embodiments, the higher likelihood of graft dysfunction may be about 20% to about 40%. In some embodiments, the higher likelihood of graft dysfunction may be about 25% to about 35%. In some embodiments, the higher likelihood of graft dysfunction may be about 25% to about 30%. In some embodiments, the higher likelihood of graft dysfunction may be about 10% to about 20%. In some embodiments, the higher likelihood of graft dysfunction may be about 15% to about 25%. In some embodiments, the higher likelihood of graft dysfunction may be about 20% to about 30%. In some embodiments, the higher likelihood of graft dysfunction may be about 30% to about 40%. In some embodiments, the higher likelihood of graft dysfunction may be about 35% to about 45%. In some embodiments, the higher likelihood of graft failure may be about 40% to about 50%.

[0041] In some embodiments, graft failure occurs after 2 years. In some embodiments, graft failure occurs after 3 years. In some embodiments, graft failure occurs after 4 years. In some embodiments, graft failure occurs after 5 years. In some embodiments, graft failure occurs after 6 years. In some embodiments, graft failure occurs after 7 years. In some embodiments, graft failure occurs after 8 years. In some embodiments, graft failure occurs after 9 years. In some embodiments, graft failure occurs within 7 years. In some embodiments, graft failure occurs within 8 years. In some embodiments, graft failure occurs within 9 years.

[0042] In some embodiments, the device may be for determining the level of fetuin A fragment and the level of urinary creatinine to assess the probability of graft failure based on the ratio and at least one other marker.

[0043] In some embodiments, the at least one other marker may include age, sex, time since transplant at enrollment, urinary albumin to creatine ratio (UACR), estimated glomerular filtration rate (eGFR) based on the creatinine and cystatin C-based CKD-EPI equation, presence of human leukocyte antigen, or any combination thereof.

[0044] In some embodiments, the assessed probability may represent at least about 70 percent (%) of the area under the receiver operating characteristic (ROC) curve (AUC). In some embodiments, the assessed probability may represent at least about 80 percent (%) of the AUC. In some embodiments, the assessed probability may represent at least about 85 percent (%) of the AUC. In some embodiments, the assessed probability may represent at least about 90 percent (%) of the AUC. In some embodiments, the assessed probability may represent at least about 95 percent (%) of the AUC.

[0045] The present disclosure may also relate to a method of correlating likelihood of graft function decline, the method may include determining levels of a first biomarker and a second biomarker in a urine sample from a kidney transplant recipient after kidney transplantation, wherein the first biomarker may be a post-translationally modified fetuin A fragment in urine fragments and the second biomarker may be urinary creatinine; determining a ratio of the level of the first biomarker to the level of the second biomarker; and correlating the ratio with likelihood of likelihood of graft function decline in the recipient over a period of about 10 years, wherein a ratio greater than about 20 ng / mg, greater than about 21 ng / mg, greater than about 22 ng / mg, greater than about 23 ng / mg, more preferably greater than about 23.5 ng / mg, and more preferably greater than about 20 ng / mg indicates a higher likelihood of graft function decline over a period of about 10 years or less.

[0046] In some embodiments, a ratio greater than about 20 ng / mg, greater than about 21 ng / mg, greater than about 22 ng / mg, greater than about 23 ng / mg, more preferably greater than about 23.5 ng / mg, and more preferably greater than about 24 ng / mg, triggers medical intervention to forestall graft failure.

[0047] In some embodiments, the higher likelihood may be greater than a lower likelihood of graft decline over a period of about 10 years or less in a second subject having a lower ratio of determined level of fetuin-A fragment to level of urinary creatinine of less than about 16 ng / mg, less than about 15.5 ng / mg, less than about 15 ng / mg, less than about 14.5 ng / mg, or less than about 14 ng / mg.

[0048] In some embodiments, graft deterioration may be indicated by a doubling of serum creatinine, the need for kidney retransplantation, or the need for dialysis.

[0049] In some embodiments, reduced graft function may be indicated by a decrease in estimated glomerular filtration rate (eGFR).

[0050] In some embodiments, interacting with a fetuin A fragment may include binding to a fetuin A fragment.

[0051] In some embodiments, interacting with urinary creatinine may include binding to urinary creatinine.

[0052] In some embodiments, at least one of the first and second reagents may comprise an antibody.

[0053] In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 25 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 25.5 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 26 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 27 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 27.5 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 28 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 28.5 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 28.7 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 28.8 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 28.9 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 29.0 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 29.1 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 29.2 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 29.3 ng / mg.

[0054] In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 30 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 34 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 35 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 37 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 38 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 40 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 50 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 60 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 65 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 70 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 71 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 72 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 73 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 75 ng / mg.

[0055] In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 80 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 85 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 90 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 91 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 92 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 93 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 94 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 90 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 95 ng / mg.

[0056] In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 13 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 12 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 11 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 10 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 9 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 8.8 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 8.5 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 8.4 ng / mg. In some embodiments, the second ratio less than about 14 ng / mg can be less than about 8.2 ng / mg.

[0057] In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 8 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 7.5 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 7.4 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 3.3 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 7.3 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 7.2 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 7.1 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 7 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 6 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 5 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 4 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 3.9 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 3.8 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 3.7 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 3.6 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 3.5 ng / mg.

[0058] In some embodiments, the higher likelihood of graft failure may be about 5% or greater. In some embodiments, the higher likelihood of graft failure may be about 8% or greater. In some embodiments, the higher likelihood of graft failure may be about 10% or greater. In some embodiments, the higher likelihood of graft failure may be about 15% or greater. In some embodiments, the higher likelihood of graft failure may be about 16% or greater. In some embodiments, the higher likelihood of graft failure may be about 20% or greater. In some embodiments, the higher likelihood of graft failure may be about 22% or greater. In some embodiments, the higher likelihood of graft failure may be about 25% or greater. In some embodiments, the higher likelihood of graft failure may be about 30% or greater. In some embodiments, the higher likelihood of graft failure may be about 32% or greater. In some embodiments, the higher likelihood of graft failure may be about 35% or greater. In some embodiments, the higher likelihood of graft failure may be about 36% or greater. In some embodiments, the higher likelihood of graft failure may be about 40% or greater. In some embodiments, the higher likelihood of graft failure may be about 45% or greater, hi some embodiments, the higher likelihood of graft failure may be about 50% or greater.

[0059] In some embodiments, the higher likelihood of graft dysfunction may be about 5% to about 50%. In some embodiments, the higher likelihood of graft dysfunction may be about 10% to about 45%. In some embodiments, the higher likelihood of graft dysfunction may be about 20% to about 40%. In some embodiments, the higher likelihood of graft dysfunction may be about 25% to about 35%. In some embodiments, the higher likelihood of graft dysfunction may be about 25% to about 30%. In some embodiments, the higher likelihood of graft dysfunction may be about 10% to about 20%. In some embodiments, the higher likelihood of graft dysfunction may be about 15% to about 25%. In some embodiments, the higher likelihood of graft dysfunction may be about 20% to about 30%. In some embodiments, the higher likelihood of graft dysfunction may be about 30% to about 40%. In some embodiments, the higher likelihood of graft dysfunction may be about 35% to about 45%. In some embodiments, the higher likelihood of graft failure may be about 40% to about 50%.

[0060] In some embodiments, graft failure occurs after 2 years. In some embodiments, graft failure occurs after 3 years. In some embodiments, graft failure occurs after 4 years. In some embodiments, graft failure occurs after 5 years. In some embodiments, graft failure occurs after 6 years. In some embodiments, graft failure occurs after 7 years. In some embodiments, graft failure occurs after 8 years. In some embodiments, graft failure occurs after 9 years. In some embodiments, graft failure occurs within 7 years. In some embodiments, graft failure occurs within 8 years. In some embodiments, graft failure occurs within 9 years.

[0061] In some embodiments, the method may further include assessing the probability of graft failure based on the ratio and at least one other marker.

[0062] In some embodiments, the at least one other marker may include age, sex, time since transplant at enrollment, urinary albumin to creatine ratio (UACR), estimated glomerular filtration rate (eGFR) based on the creatinine and cystatin C-based CKD-EPI equation, presence of human leukocyte antigen, or any combination thereof.

[0063] In some embodiments, the assessed probability may represent at least about 70 percent (%) of the area under the receiver operating characteristic (ROC) curve (AUC). In some embodiments, the assessed probability may represent at least about 80 percent (%) of the AUC. In some embodiments, the assessed probability may represent at least about 85 percent (%) of the AUC. In some embodiments, the assessed probability may represent at least about 90 percent (%) of the AUC. In some embodiments, the assessed probability may represent at least about 95 percent (%) of the AUC. [Brief explanation of the drawings]

[0064] [Figure 1] 1 shows Kaplan-Meier survival curves for kidney transplant recipients (KTRs) with graft failure censored at death in the primary set, according to non-limiting embodiments. [Figure 2A] 1 shows Kaplan-Meier curves of KTR in the primary set with (a) non-proteinuria by uPTM-FetA / UCr risk classification, according to non-limiting embodiments. [Figure 2B] (b) Kaplan-Meier curves of KTR in the primary set with proteinuria by uPTM-FetA / UCr risk classification, according to non-limiting embodiments. [Figure 3] 1 shows Kaplan-Meier survival curves for KTRs with graft failure censored at death in the validation set, according to non-limiting embodiments. [Figure 4A]1 shows Kaplan-Meier curves of KTR in the validation set with (a) non-proteinuria by uPTM-FetA / UCr risk classification, according to non-limiting embodiments. [Figure 4B] 1 shows Kaplan-Meier curves of KTR in the validation set with (b) proteinuria by uPTM-FetA / UCr risk classification, according to non-limiting embodiments. [Figure 5] 1 shows Kaplan-Meier survival curves for 635 KTRs with graft failure censored at death, according to non-limiting embodiments. [Figure 6A] 1 shows Kaplan-Meier curves of KTR in the 24-hour set with (a) non-proteinuria by uPTM-FetA / UCr risk classification, according to non-limiting embodiments. [Figure 6B] (b) Kaplan-Meier curves of KTR in the 24-hour set with proteinuria by uPTM-FetA / UCr risk classification, according to non-limiting embodiments. [Figure 7] 1 shows the receiver operating characteristic (ROC) curve for uPTM-FetA / UCr and the corresponding Kaplan-Meier curve for the marker combo model of KTR with graft failure over 2 years using spot urine, according to non-limiting embodiments. [Figure 8] 1 shows receiver operating characteristic (ROC) curves for the combination of uPTM-FetA / UCr, age, sex, time, and UACR, and the corresponding Kaplan-Meier curves of the marker combo model for KTRs with graft failure over 2 years using spot urine, according to non-limiting embodiments. [Figure 9] FIG. 1 shows receiver operating characteristic (ROC) curves of uPTM-FetA / UCr, age, sex, time, UACR, and eGFR, and the corresponding Kaplan-Meier curves of the marker combo model, for KTRs with graft failure over 2 years using spot urine, according to non-limiting embodiments. [Figure 10]1 shows receiver operating characteristic (ROC) curves of uPTM-FetA / UCr, age, sex, time, UACR, eGFR, and presence of human leukocyte antigen class II antibodies (HLA II) and corresponding Kaplan-Meier curves of marker combo models for KTRs with graft failure over 2 years using spot urine, according to non-limiting embodiments. [Figure 11] 1 shows the receiver operating characteristic (ROC) curve for uPTM-FetA / UCr and the corresponding Kaplan-Meier curve for the marker combo model of KTRs with graft failure over 5 years using spot urine, according to non-limiting embodiments. [Figure 12] 1 shows receiver operating characteristic (ROC) curves for the combination of uPTM-FetA / UCr, age, sex, time, and UACR, and the corresponding Kaplan-Meier curves of the marker combo model for KTRs with graft failure over 5 years using spot urine, according to non-limiting embodiments. [Figure 13] FIG. 1 shows receiver operating characteristic (ROC) curves of uPTM-FetA / UCr, age, sex, time, UACR, and eGFR, and corresponding Kaplan-Meier curves of marker combo models for KTRs with graft failure over 5 years using spot urine, according to non-limiting embodiments. [Figure 14] 1 shows receiver operating characteristic (ROC) curves of uPTM-FetA / UCr, age, sex, time, UACR, eGFR, and presence of human leukocyte antigen class II antibodies (HLA II) and corresponding Kaplan-Meier curves of marker combo models for KTRs with 5-year graft failure using spot urine, according to non-limiting embodiments. [Figure 15] 1 shows a flowchart of a study population selection, according to a non-limiting embodiment. [Figure 16] 1 shows a scatter plot and visual representation of the correlation between 24-hour uPTM-FetA excretion and serum creatine levels, according to a non-limiting embodiment. [Figure 17] 1 shows a scatter plot and visual representation of the correlation between 24-hour uPTM-FetA excretion and eGFR, according to a non-limiting embodiment. [Figure 18] 1 shows a scatter plot and visual representation of the correlation between 24-hour uPTM-FetA excretion and 24-hour albumin excretion, according to a non-limiting embodiment. [Figure 19] 1 shows a scatter plot and visual representation of the correlation between 24-hour uPTM-FetA excretion and 24-hour urinary protein, according to a non-limiting embodiment. [Figure 20] 1 shows Kaplan-Meier curves for graft failure censored at death below and above the median 24-hour urinary excretion of post-translationally modified fetuin-A, according to non-limiting embodiments. [Figure 21] 1 shows Kaplan-Meier curves for graft decline censored at death below and above the median 24-hour urinary excretion of post-translationally modified fetuin-A, according to non-limiting embodiments. [Figure 22] 1 shows Kaplan-Meier curves for all-cause mortality below and above the median 24-hour urinary excretion of post-translationally modified fetuin-A, according to non-limiting embodiments. [Figure 23] 1 shows a flowchart of study participant selection, according to a non-limiting embodiment. [Figure 24] 1 shows a Kaplan-Meier analysis of death-censored graft decline survival per tertile of 24-hour uC-FetA excretion, according to non-limiting embodiments. [Figure 25] 1 shows a forest plot of the association between uC-FetA and death-censored graft function decline in subgroups, according to non-limiting embodiments. [Figure 26] 1 shows a Kaplan-Meier analysis of patient survival per tertile of 24-hour uC-FetA excretion, according to non-limiting embodiments. [Figure 27] 1 shows a calibration curve for Human uPTM3-DKD ELISA according to a non-limiting embodiment (calibration range is 7.813 to 500 ng / mL). DETAILED DESCRIPTION OF THE INVENTION

[0065] The present disclosure is based on the unexpected discovery that a urinary protein and its fragment, urinary post-translationally modified fetuin A fragment (uPTM-FetA), are differentially represented in kidney transplant recipients (KTRs) who are more likely to experience graft failure. Therefore, these protein molecules are useful markers for diagnosing graft failure at various stages, including early stages. Therefore, uPTM-FetA has been identified as a biomarker for acute kidney injury and, by extension, proposed as a biomarker for early detection of renal function decline.

[0066] Kidney transplantation remains the most preferred treatment for ESKD to date because it offers a better quality of life and is the most cost-effective option compared with other renal replacement therapies. However, despite this success, kidney transplant recipients (KTRs) may remain at risk for graft loss and potentially morbidity and early mortality, primarily due to declining renal graft function. From the patient's perspective, kidney graft survival is perceived as more important than life itself, and patients would rather die than return to dialysis. Therefore, more efforts must be made to detect graft function loss early so that appropriate treatment can be administered and graft failure can be prevented. In the current clinical environment, routine assessment of graft function is based on serum creatinine and proteinuria. Renal biopsy is considered when serum creatinine is significantly elevated or proteinuria is present. Unfortunately, these assessments are imperfect, especially with regard to predicting graft prognosis. Therefore, there is a need for biomarkers that can be used to identify KTRs at risk for graft function decline and subsequent graft failure, including alternative noninvasive markers that can independently identify patients at risk for graft function decline and subsequent graft failure.

[0067] As used herein, the term "medical intervention" in the context of declining renal graft function in a KTR refers to treating the recipient in an attempt to stabilize, slow, delay, or prevent the decline, which may result in a decrease in the rate of decline in the recipient. Various types of medical interventions can be performed. Examples of medical interventions include induction diets, restricted diets, limiting sodium and other ion intake, reducing or avoiding alcohol consumption, reducing or avoiding tobacco smoking or consumption of related products, taking various medications, weight management, kidney transplantation or retransplantation, dialysis, other medically recognized treatments, and any combination thereof.

[0068] As used herein, the term "average fetuin A fragment excretion" refers to the amount of fetuin A fragment, such as fetuin A containing a binding peptide, excreted in urine averaged over a given period of time. For example, the average fetuin A fragment excretion can be averaged over a 2-48 hour period or a shorter period of time.

[0069] Thus, the present disclosure relates to diagnostic methods using biomarkers, which can be proteins that can be considered proteins or fragments of proteins. Urine samples, serum samples, or both urine and serum samples can be collected from a subject, and the urinary level, serum level, or both levels of the biomarker can be determined via various methods, such as, for example, mass spectrometry and immunoassay.

[0070] When a biomarker contains a single protein molecule, its level in a subject can be compared with a reference point to determine the corresponding signs or likelihood of a target condition. A reference point representing the level of the same biomarker, such as the level of a biomarker in normal subjects without substantial decline in renal graft function in a KTR, can be determined based on the representative level of the biomarker in a group of KTRs. For example, the reference point can be the midpoint between the average levels of these two groups. A biomarker level higher than the reference point indicates a target condition.

[0071] There is a great clinical need for novel markers to predict renal function decline in patients with ESKD or KTR. Urinary post-translationally modified fetuin A fragments (uPTM-FetA) may be a biomarker for indicating possible renal function decline on renal endpoints and can predict renal function decline in patients with ESKD or KTR.

[0072] Fetuin A (also known as alpha-2-Heremans-Schmidt glycoprotein) is a heterodimeric glycoprotein with a 367-amino acid sequence, including an 18-amino acid signaling protein, a 282-amino acid A chain, a 40-amino acid connecting peptide, and a 27-amino acid B chain that undergoes various post-translational modifications before secretion outside of the producing cell. Fetuin A, including urinary post-translationally modified fetuin A fragments (uPTM-FetA) (also known as alpha-2-Heremans-Schmidt glycoprotein), has properties suggesting that it may serve as an alternative, independent, non-invasive marker. Fetuin A is elevated in the urine of patients with acute kidney injury (AKI), and recent scientific evidence suggests that it is associated with the degree of interstitial fibrosis / tubular atrophy (IFTA). The occurrence of AKI and the presence of IFTA after kidney transplantation are known to be associated with unfavorable long-term graft outcomes. Urinary fetuin-A levels are also elevated in patients with chronic kidney diseases, such as autosomal dominant polycystic kidney disease and focal segmental glomerulosclerosis. Furthermore, urinary fetuin-A levels have previously been shown to be associated with decreased renal function in patients with chronic kidney disease, including diabetic nephropathy, and this protein has been proposed as a biomarker for early detection of these diseases. However, no studies have been conducted to measure urinary fetuin-A levels in the KTR population and to investigate its plausible association with graft outcomes.

[0073] In some embodiments, urinary post-translationally modified fetuin A fragment (uPTM-FetA) can be used as a biomarker for acute kidney injury and, by extension, has been proposed as a biomarker for early detection of declining kidney function. In some embodiments, uPTM-FetA protein can serve as a marker of graft decline in KTRs. For example, levels of uPTM-FetA in stable outpatient KTRs can be used to determine the likelihood of a higher likelihood of graft decline over time, such as up to about 10 years. In some embodiments, the primary endpoint may be graft decline censored at death, i.e., the need for retransplantation or (re)initiation of dialysis, and the secondary endpoint was all-cause mortality.

[0074] Because of the potential pathophysiological role of urinary fetuin-A in the development of renal function decline, the association between urinary fetuin-A or its fragments and graft function decline can be used to determine the likelihood of graft function decline over time.

[0075] Thus, in some embodiments, a method of preparing an assay, for example to correlate the likelihood of graft failure in a kidney transplant recipient, includes providing a first solution comprising a urine sample from the kidney transplant recipient after kidney transplantation and a first reagent for interacting with post-translationally modified fetuin in the urine fragment to determine the level of fetuin A fragment in the urine sample; providing a second solution comprising the urine sample and a second reagent for interacting with urinary creatinine to determine the level of urinary creatinine in the urine sample; and comparing the determined level of fetuin A fragment and the determined level of urinary creatinine. and correlating a ratio of the determined level of fetuin-A fragment to the determined level of urinary creatinine with a likelihood of graft failure in the recipient, depending on the ratio, wherein a ratio greater than about 20 ng / mg, greater than about 21 ng / mg, greater than about 22 ng / mg, greater than about 23 ng / mg, more preferably greater than about 23.5 ng / mg, and more preferably greater than about 20 ng / mg indicates a higher likelihood of graft failure over a period of about 10 years or less, and wherein the first solution and the second solution are the same solution or different solutions from each other.

[0076] Furthermore, in some embodiments, an assay kit for determining the likelihood of progressive renal function decline may include a first solution comprising a first reagent for interacting with post-translationally modified fetuin-A fragments in urinary fragments to indicate the level of fetuin-A fragments in a urine sample from a kidney transplant recipient after kidney transplantation; a second solution comprising a second reagent for interacting with urinary creatinine to indicate the level of urinary creatinine in the urine sample; and a device for determining the level of fetuin-A fragments and the level of urinary creatinine in the urine sample and determining a ratio of the determined level of fetuin-A fragments to the determined level of urinary creatinine, wherein a ratio greater than about 20 ng / mg, greater than about 21 ng / mg, greater than about 22 ng / mg, greater than about 23 ng / mg, more preferably greater than about 23.5 ng / mg, more preferably greater than about 24 ng / mg indicates a higher likelihood of graft function decline over a period of about 10 years or less, and wherein the first solution and the second solution are the same solution or different solutions from each other.

[0077] Further, in some embodiments, a method of correlating the likelihood of graft function decline may include determining levels of a first biomarker and a second biomarker in a urine sample from a kidney transplant recipient after kidney transplant, where the first biomarker can be a post-translationally modified fetuin A fragment in urine and the second biomarker can be urinary creatinine; determining a ratio of the level of the first biomarker to the level of the second biomarker; and correlating the ratio with the likelihood of graft function decline in the recipient over a period of about 10 years, where a ratio greater than about 20 ng / mg, greater than about 21 ng / mg, greater than about 22 ng / mg, greater than about 23 ng / mg, more preferably greater than about 23.5 ng / mg, and more preferably greater than about 24 ng / mg indicates a higher likelihood of graft function decline over a period of about 10 years or less.

[0078] In some embodiments, a ratio greater than about 20 ng / mg, greater than about 21 ng / mg, greater than about 22 ng / mg, greater than about 23 ng / mg, more preferably greater than about 23.5 ng / mg, and more preferably greater than about 24 ng / mg, triggers medical intervention to forestall graft failure.

[0079] In some embodiments, the higher likelihood may be greater than a lower likelihood of graft decline over a period of about 10 years or less in a second subject having a lower ratio of determined level of fetuin-A fragment to level of urinary creatinine of less than about 16 ng / mg, less than about 15.5 ng / mg, less than about 15 ng / mg, less than about 14.5 ng / mg, or less than about 14 ng / mg.

[0080] In some embodiments, graft deterioration may be indicated by a doubling of serum creatinine, the need for kidney retransplantation, or the need for dialysis.

[0081] In some embodiments, reduced graft function may be indicated by a decrease in estimated glomerular filtration rate (eGFR).

[0082] In some embodiments, interacting with a fetuin A fragment may include binding to a fetuin A fragment.

[0083] In some embodiments, interacting with urinary creatinine may include binding to urinary creatinine.

[0084] In some embodiments, at least one of the first and second reagents may comprise an antibody.

[0085] In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 25 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 25.5 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 26 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 27 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 27.5 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 28 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 28.5 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 28.7 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 28.8 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 28.9 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 29.0 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 29.1 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 29.2 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 29.3 ng / mg.

[0086] In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 30 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 34 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 35 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 37 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 38 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 40 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 50 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 60 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 65 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 70 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 71 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 72 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 73 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 75 ng / mg.

[0087] In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 80 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 85 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 90 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 91 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 92 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 93 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 94 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 90 ng / mg. In some embodiments, a ratio greater than about 20 ng / mg may be greater than about 95 ng / mg.

[0088] In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 13 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 12 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 11 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 10 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 9 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 8.8 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 8.5 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 8.4 ng / mg. In some embodiments, the second ratio less than about 14 ng / mg can be less than about 8.2 ng / mg.

[0089] In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 8 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 7.5 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 7.4 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 3.3 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 7.3 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 7.2 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 7.1 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 7 ng / mg. In some embodiments, the second ratio less than about 14 ng / mg can be less than about 6 ng / mg.

[0090] In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 5 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 4 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 3.9 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 3.8 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 3.7 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 3.6 ng / mg. In some embodiments, the second ratio lower than about 14 ng / mg can be lower than about 3.5 ng / mg.

[0091] In some embodiments, the higher likelihood of graft failure may be about 5% or greater. In some embodiments, the higher likelihood of graft failure may be about 8% or greater. In some embodiments, the higher likelihood of graft failure may be about 10% or greater. In some embodiments, the higher likelihood of graft failure may be about 15% or greater. In some embodiments, the higher likelihood of graft failure may be about 16% or greater.

[0092] In some embodiments, the higher likelihood of graft failure may be about 20% or greater. In some embodiments, the higher likelihood of graft failure may be about 22% or greater. In some embodiments, the higher likelihood of graft failure may be about 25% or greater. In some embodiments, the higher likelihood of graft failure may be about 30% or greater. In some embodiments, the higher likelihood of graft failure may be about 32% or greater. In some embodiments, the higher likelihood of graft failure may be about 35% or greater. In some embodiments, the higher likelihood of graft failure may be about 36% or greater. In some embodiments, the higher likelihood of graft failure may be about 40% or greater. In some embodiments, the higher likelihood of graft failure may be about 45% or greater. In some embodiments, the higher likelihood of graft failure may be about 50% or greater.

[0093] In some embodiments, the higher likelihood of graft dysfunction may be about 5% to about 50%. In some embodiments, the higher likelihood of graft dysfunction may be about 10% to about 45%. In some embodiments, the higher likelihood of graft dysfunction may be about 20% to about 40%. In some embodiments, the higher likelihood of graft dysfunction may be about 25% to about 35%. In some embodiments, the higher likelihood of graft dysfunction may be about 25% to about 30%. In some embodiments, the higher likelihood of graft dysfunction may be about 10% to about 20%. In some embodiments, the higher likelihood of graft dysfunction may be about 15% to about 25%. In some embodiments, the higher likelihood of graft dysfunction may be about 20% to about 30%. In some embodiments, the higher likelihood of graft dysfunction may be about 30% to about 40%. In some embodiments, the higher likelihood of graft dysfunction may be about 35% to about 45%. In some embodiments, the higher likelihood of graft failure may be about 40% to about 50%.

[0094] In some embodiments, graft failure occurs after 2 years. In some embodiments, graft failure occurs after 3 years. In some embodiments, graft failure occurs after 4 years. In some embodiments, graft failure occurs after 5 years. In some embodiments, graft failure occurs after 6 years. In some embodiments, graft failure occurs after 7 years. In some embodiments, graft failure occurs after 8 years. In some embodiments, graft failure occurs after 9 years. In some embodiments, graft failure occurs within 7 years. In some embodiments, graft failure occurs within 8 years. In some embodiments, graft failure occurs within 9 years.

[0095] In some embodiments, the method may further include assessing the probability of graft failure based on the ratio and at least one other marker.

[0096] In some embodiments, the at least one other marker may include age, sex, time since transplant at enrollment, urinary albumin to creatine ratio (UACR), estimated glomerular filtration rate (eGFR) based on the creatinine and cystatin C-based CKD-EPI equation, presence of human leukocyte antigen, or any combination thereof.

[0097] In some embodiments, the assessed probability may represent at least about 70 percent (%) of the area under the receiver operating characteristic (ROC) curve (AUC). In some embodiments, the assessed probability may represent at least about 80 percent (%) of the AUC. In some embodiments, the assessed probability may represent at least about 85 percent (%) of the AUC. In some embodiments, the assessed probability may represent at least about 90 percent (%) of the AUC. In some embodiments, the assessed probability may represent at least about 95 percent (%) of the AUC.

[0098] In some embodiments, interacting with the fetuin A fragment comprises binding to fetuin A. In some embodiments, the reagent comprises an antibody.

[0099] uPTM-FetA excretion correlates with renal function and is also associated with increased markers of proximal tubular injury and 24-hour urinary protein excretion. Importantly, uPTM-FetA excretion is independently associated with graft decline in stable KTRs, especially in patients with relatively good renal function.

[0100] In some embodiments, in KTRs, shorter time post-transplant, surviving donors, older donor age, use of growth inhibitors, and lower AST levels may be independently associated with increased uPTM-FetA excretion. In some embodiments, urinary L-FABP excretion, a marker of proximal tubule injury, may also be independently associated with uPTM-FetA excretion. In some embodiments, increased uPTM-FetA excretion may be associated with an increased risk of graft dysfunction, even after adjusting for potential confounding factors. In healthy adults, the kidney does not express fetuin A. However, upon injury, proximal tubule epithelial cells (PTECs) can express and release fetuin A to the luminal side of the tubule. In cisplatin-induced and ischemia / reperfusion-induced AKI rat models, urinary fetuin A can be predominantly present in the urinary exosome fraction, rather than from the non-exosome fraction. PTECs can locally produce fetuin A under hypoxic conditions after stimulation from hypoxia-inducible transcription factors. The presence of fetuin A within the proximal tubule may help protect the kidney from hypoxia-induced renal inflammation by preventing the shift of macrophages to the proinflammatory M1 macrophage and from hypoxia-induced fibrosis by antagonizing TGFβ signaling.

[0101] The human precursor protein of fetuin A consists of three parts: the A chain, the connecting peptide, and the B chain, which are 321, 40, and 27 amino acids long, respectively. The connecting peptide of the precursor is removed by post-translational modification (i.e., limited proteolysis), after which only the A and B chains form the active fetuin A protein. The monoclonal antibody in the ELISA kit can detect the connecting peptide-containing fetuin A. Therefore, the uPTM fetuin A detected by this ELISA kit may be inactive fetuin A without protective effects on renal function, and elevated uPTM-FetA has been associated with the risk of reduced eGFR in patients. In addition to local production, fetuin A in urine can also originate from the circulation. Fetuin A is a negatively charged molecule with a molecular weight of approximately 60 kilodaltons, similar to that of albumin. Under physiological conditions, a selective amount of albumin can cross the glomerular filtration barrier (GFB), where it is reabsorbed by proximal tubular epithelial cells (PTEC) within the tubular compartment. Because fetuin-A and albumin have similar properties, comparable processing by the kidney can be expected. Fetuin-A can be absorbed into the proximal tubule of healthy rats by megalin-mediated endocytosis, primarily in the S1 segment and, to a lesser extent, in the S2 segment. Under conditions of accumulated glomerular damage, the GFB is disrupted, resulting in excessive leakage of different plasma proteins into the tubular compartment. This increases the amount of filtered proteins that the proximal tubule must reabsorb. Because the reabsorption capacity of PTEC is not infinite, saturation of this mechanism leads to the presence of proteins from the circulation in the urine.The filtered proteins can then cause damage within the tubules through various mechanisms: by clogging the tubular lumen with protein casts, by causing energy depletion and lysosomal rupture due to the excessive protein overload that PTECs must reabsorb, by triggering PTECs to release inflammatory and profibrotic cytokines, by causing the intracellular accumulation of lipidated proteins that induce tubular apoptosis, and by activating the complement system, causing direct damage to PTECs. When this occurs, the ability of PTECs to locally produce fetuin A and reabsorb filtered fetuin A may be impaired. Because the excretion level of uPTM-FetA was significantly associated with an increased risk of graft failure, even after adjusting for potential confounding factors, including urinary protein excretion, the origin of uPTM-FetA may be due to local production by PTECs rather than an impaired reabsorption process. In sensitivity analyses, uPTM-FetA may not be associated with decreased graft function in patients with eGFR <30 ml / min / 1.73 m2. Fetuin A from the circulation may also end up in the urine if there is accumulated damage to the kidney. Therefore, uPTM-FetA measured in this patient subgroup may consist of locally produced fetuin A and circulating fetuin A. Circulating fetuin A is not associated with renal function.

[0102] Both acute and chronic kidney injury are closely associated with the development of hypoxia. Among all structures within the kidney, PTECs are the most vulnerable due to their high activity and oxygen demand. Because hypoxia plays a major role in the progression of kidney disease and can be present relatively early in kidney injury, even before the onset of structural damage, early identification of this condition may be beneficial to prevent further deterioration. In both cisplatin-induced and ischemia / reperfusion-induced AKI, urinary fetuin-A excretion increased before the rapid rise in serum creatinine. Furthermore, fetuin-A deposition in PTECs increased before morphologically present structural damage to the renal tubules. In type 2 diabetes patients, urinary fetuin-A may indicate the progression of renal function decline earlier than albuminuria. Since one of the most important requirements for a biomarker is that it reflects the underlying pathophysiology of the disease, in some embodiments, measurement of urinary fetuin A may offer additional advantages as a biomarker for early detection of graft damage in KTR over currently used parameters such as serum creatinine or proteinuria.

[0103] In some embodiments, correlation of fetuin-A with at least one other renal biomarker can be an indicator of renal function or a decline in renal function. For example, correlation of fetuin-A with creatinine can be such an indicator. Renal function can be determined by the level of filtration that occurs. Filtration is accomplished by tiny blood vessels within the kidney called "glomeruli." Overall renal function is therefore referred to as the "glomerular filtration rate" (GFR), which is measured as the amount of blood filtered per minute (milliliters / minute). GFR can vary depending on other variables, such as age and gender. GFR can be estimated using a formula that takes into account age, gender, and a blood test called creatinine (eGFR). Renal failure is most often seen when creatinine levels are high, indicating declining renal function. Creatinine is a molecule produced in muscles. Healthy kidneys remove creatinine from the bloodstream and excrete it in urine. Higher creatinine in the blood is a sign that the kidneys are not cleaning blood as well as they should. Therefore, correlating fetuin-A with GFR or creatinine may be a relatively reliable indicator of declining renal function in KTR.

[0104] Assay In some embodiments, various methods, reagents, devices, and kits can be used to measure levels of fetuin A or fetuin A-based fragments in various media, such as urine, serum, and other bodily fluids. In some embodiments, enzyme-linked immunosorbent assays (ELISAs) can be used to measure fetuin A levels. For example, ELISAs can be used to quantitatively measure fetuin A with specific post-translational modifications (PTMs) in human urine and should be performed in a competent clinical laboratory by a certified medical professional, such as a medical technician. For example, a calibrator or unknown urine sample is mixed with an antibody that has binding affinity for a portion of fetuin A, such as a monoclonal antibody (mAb) against fetuin A with a specific PTM, and then incubated in a microplate pre-bound with fetuin A or a segment thereof, such as fetuin A with a specific PTM. The monoclonal antibody recognizes the fetuin A in the calibrator or unknown sample in competition with the antibody in the microplate well. After incubation, in some embodiments, a signal-generating molecule, such as a signal molecule-tagged antibody, can be used. In some embodiments, a horseradish peroxide (HRP)-conjugated secondary antibody is added, followed by incubation with 3,3',5,5'-tetramethylbenzidine (TMB) substrate. Their relative reactivities are determined by absorbance measurements at 450 nanometers (nm) and plotted against a predetermined specific PTM fetuin A calibration curve.

[0105] In some embodiments, a method for correlating the likelihood of graft failure in a kidney transplant recipient includes obtaining a solution containing a urine sample from the kidney transplant recipient after kidney transplantation and a reagent for interacting with urinary binding peptide-containing fetuin A fragments, measuring the average fetuin A fragment excretion into the urine from the solution, and correlating the measured average fetuin A fragment excretion with the likelihood of graft failure in the recipient, wherein a measured average fetuin A fragment excretion higher than about 0.8 micrograms per hour (μg / h) indicates a higher likelihood of graft failure over a period of about 10 years or less.

[0106] In some embodiments, a method of preparing an assay includes providing a solution containing a urine sample from a kidney transplant recipient after kidney transplantation and a reagent for interacting with urinary binding peptide-containing fetuin A fragments; measuring the average fetuin A fragment excretion into urine from the solution; and correlating the measured average fetuin A fragment excretion with a likelihood of graft failure in the recipient, wherein a measured average fetuin A fragment excretion higher than about 0.8 micrograms per hour (μg / h) indicates a higher likelihood of graft failure over a period of about 10 years or less.

[0107] In some embodiments, an assay kit for determining the likelihood of graft failure in a transplanted kidney can include a solution containing a reagent at a concentration to interact with the amount of urinary binding peptide-containing fetuin-A fragments in a urine sample from a kidney transplant recipient. A device can be provided to measure the average urinary excretion of fetuin-A fragments from the solution and correlate the measured average fetuin-A fragment excretion with the likelihood of graft failure in the recipient, where a measured average fetuin-A fragment excretion greater than about 0.8 micrograms per hour (μg / h) indicates a higher likelihood of graft failure over a period of about 10 years or less.

[0108] In some embodiments, a method for correlating the likelihood of graft failure in a kidney transplant recipient includes obtaining a solution containing a urine sample from the kidney transplant recipient after kidney transplantation and a reagent for interacting with urinary binding peptide-containing fetuin A fragments, measuring the average fetuin A fragment excretion into the urine from the solution, and correlating the measured average fetuin A fragment excretion with the likelihood of graft failure in the recipient, wherein a measured average fetuin A fragment excretion higher than about 0.8 micrograms per hour (μg / h) indicates a higher likelihood of graft failure over a period of about 10 years or less.

[0109] In some embodiments, a method of preparing an assay includes providing a solution containing a urine sample from a kidney transplant recipient after kidney transplantation and a reagent for interacting with urinary binding peptide-containing fetuin A fragments; measuring the average fetuin A fragment excretion into urine from the solution; and correlating the measured average fetuin A fragment excretion with a likelihood of graft failure in the recipient, wherein a measured average fetuin A fragment excretion higher than about 0.8 micrograms per hour (μg / h) indicates a higher likelihood of graft failure over a period of about 10 years or less.

[0110] In some embodiments, an assay kit for determining the likelihood of graft failure in a transplanted kidney can include a solution containing a reagent at a concentration to interact with the amount of urinary binding peptide-containing fetuin-A fragments in a urine sample from a kidney transplant recipient. A device can be provided to measure the average urinary excretion of fetuin-A fragments from the solution and correlate the measured average fetuin-A fragment excretion with the likelihood of graft failure in the recipient, where a measured average fetuin-A fragment excretion greater than about 0.8 micrograms per hour (μg / h) indicates a higher likelihood of graft failure over a period of about 10 years or less.

[0111] The present disclosure relates to diagnostic methods using biomarkers, which can be proteins that can be considered proteins or fragments of proteins. Urine samples, serum samples, or both urine and serum samples can be collected from a subject, and the urinary level, serum level, or both levels of the biomarker can be determined via various methods, such as, for example, mass spectrometry and immunoassay.

[0112] When a biomarker contains a single protein molecule, its level in a subject can be compared with a reference point to determine the corresponding signs or likelihood of a target condition. A reference point representing the level of the same biomarker, such as the level of a biomarker in normal subjects without substantial decline in renal graft function in a KTR, can be determined based on the representative level of the biomarker in a group of KTRs. For example, the reference point can be the midpoint between the average levels of these two groups. A biomarker level higher than the reference point indicates a target condition.

[0113] Fetuin A (also known as alpha-2-Heremans-Schmidt glycoprotein), such as urinary binding peptide-containing fetuin A (uC-FetA), has properties suggesting that it could serve as such a surrogate, independent, noninvasive marker. Fetuin A is elevated in the urine of patients with acute kidney injury (AKI), and recent scientific evidence suggests that it is associated with the degree of interstitial fibrosis / tubular atrophy (IFTA). The occurrence of AKI and the presence of IFTA after kidney transplantation are known to be associated with poor long-term graft outcomes. Urinary fetuin A has also been elevated in patients with chronic kidney diseases, such as autosomal dominant polycystic kidney disease and focal segmental glomerulosclerosis. Furthermore, urinary fetuin A has previously been shown to be associated with decreased renal function in patients with chronic kidney disease, including diabetic nephropathy, and this protein has been proposed as a biomarker for early detection of these diseases. However, no studies have been conducted to measure urinary fetuin-A in the KTR population and investigate its plausible association with graft outcome.

[0114] In some embodiments, urinary binding peptide-containing fetuin A (uC-FetA) can be used as a biomarker for acute kidney injury and, by extension, has been proposed as a biomarker for early detection of renal function decline. In some embodiments, uC-FetA protein can serve as a marker of graft decline in KTRs. For example, levels of uC-FetA in stable outpatient KTRs can be used to determine the likelihood of a higher likelihood of graft decline over time, such as up to about 10 years. In some embodiments, the primary endpoint may be graft decline censored at death, i.e., the need for retransplantation or (re)initiation of dialysis, and the secondary endpoint was all-cause mortality.

[0115] Because of the potential pathophysiological role of urinary fetuin-A in the development of renal function decline, the association between urinary fetuin-A or its fragments and graft function decline can be used to determine the likelihood of graft function decline over time.

[0116] Thus, in some embodiments, a method for correlating the likelihood of graft failure in a kidney transplant recipient includes obtaining a solution containing a urine sample from the kidney transplant recipient after kidney transplantation and a reagent for interacting with urinary binding peptide-containing fetuin A fragments, measuring the average fetuin A fragment excretion into the urine from the solution, and correlating the measured average fetuin A fragment excretion with the likelihood of graft failure in the recipient, wherein a measured average fetuin A fragment excretion higher than about 0.8 micrograms per hour (μg / h) indicates a higher likelihood of graft failure over a period of about 10 years or less.

[0117] In some embodiments, a method of preparing an assay includes providing a solution containing a urine sample from a kidney transplant recipient after kidney transplantation and a reagent for interacting with urinary binding peptide-containing fetuin A fragments; measuring the average fetuin A fragment excretion into urine from the solution; and correlating the measured average fetuin A fragment excretion with a likelihood of graft failure in the recipient, wherein a measured average fetuin A fragment excretion higher than about 0.8 micrograms per hour (μg / h) indicates a higher likelihood of graft failure over a period of about 10 years or less.

[0118] In some embodiments, an assay kit for determining the likelihood of graft failure in a transplanted kidney can include a solution containing a reagent at a concentration to interact with the amount of urinary binding peptide-containing fetuin-A fragments in a urine sample from a kidney transplant recipient. A device such as an ELISA reader can be provided to measure the average urinary excretion of fetuin-A fragments from the solution and correlate the measured average fetuin-A fragment excretion with the likelihood of graft failure in the recipient, with a measured average fetuin-A fragment excretion greater than about 0.8 micrograms per hour (μg / h) indicating a higher likelihood of graft failure over a period of about 10 years or less.

[0119] In some embodiments, excretion rates greater than about 0.8 μg / h may trigger medical intervention to forestall graft failure.

[0120] In some embodiments, graft deterioration may be indicated by a doubling of serum creatinine, the need for kidney retransplantation, or the need for dialysis.

[0121] In some embodiments, the recipient is required to maintain a urine flow rate of approximately 25 milliliters per minute per 1.73 square meters (mL / min / 1.73 m) at a time near the time of urine sample collection. 2 ) or higher.

[0122] In some embodiments, the average amount of fetuin A fragment excreted in urine may be averaged over about 2 hours or more. In some embodiments, the average amount of fetuin A fragment excreted in urine may be averaged over about 4 hours or more. In some embodiments, the average amount of fetuin A fragment excreted in urine may be averaged over about 6 hours or more. In some embodiments, the average amount of fetuin A fragment excreted in urine may be averaged over about 12 hours or more. In some embodiments, the average amount of fetuin A fragment excreted in urine may be averaged over about 24 hours or more. In some embodiments, the average amount of fetuin A fragment excreted in urine may be averaged over about 36 hours or more. In some embodiments, the average amount of fetuin A fragment excreted in urine may be averaged over about 48 hours or more.

[0123] In some embodiments, a urine sample can be collected from a kidney transplant recipient at least about 1 day after kidney transplant. In some embodiments, a urine sample can be collected from a kidney transplant recipient at least about 2 days after kidney transplant. In some embodiments, a urine sample can be collected from a kidney transplant recipient at least about 1 week after kidney transplant. In some embodiments, a urine sample can be collected from a kidney transplant recipient at least about 2 weeks after kidney transplant. In some embodiments, a urine sample can be collected from a kidney transplant recipient at least about 1 month after kidney transplant. In some embodiments, a urine sample can be collected from a kidney transplant recipient at least about 3 months after kidney transplant. In some embodiments, a urine sample can be collected from a kidney transplant recipient at least about 6 months after kidney transplant. In some embodiments, a urine sample can be collected from a kidney transplant recipient at least about 12 months after kidney transplant. In some embodiments, a urine sample can be collected from a kidney transplant recipient at least about 24 months after kidney transplant.

[0124] In some embodiments, an average fetuin-A fragment excretion rate greater than about 0.8 μg / h indicates a higher likelihood of graft function decline in the transplanted kidney over a period of about 9 years or less. In some embodiments, an average fetuin-A fragment excretion rate greater than about 0.8 μg / h indicates a higher likelihood of graft function decline in the transplanted kidney over a period of about 7 years or less. In some embodiments, an average fetuin-A fragment excretion rate greater than about 0.8 μg / h indicates a higher likelihood of graft function decline in the transplanted kidney over a period of about 5 years or less. In some embodiments, an average fetuin-A fragment excretion rate greater than about 0.8 μg / h indicates a higher likelihood of graft function decline in the transplanted kidney over a period of about 3 years or less. In some embodiments, an average fetuin-A fragment excretion rate greater than about 0.8 μg / h indicates a higher likelihood of graft function decline in the transplanted kidney over a period of about 1 year or less.

[0125] In some embodiments, an average fetuin-A fragment excretion rate greater than about 0.8 μg / h can be greater than about 0.9 μg / h. In some embodiments, an average fetuin-A fragment excretion rate greater than about 0.8 μg / h can be greater than about 1 μg / h. In some embodiments, an average fetuin-A fragment excretion rate greater than about 0.8 μg / h can be greater than about 2 μg / h. In some embodiments, an average fetuin-A fragment excretion rate greater than about 0.8 μg / h can be greater than about 2.1 μg / h. In some embodiments, an average fetuin-A fragment excretion rate greater than about 0.8 μg / h can be greater than about 2.2 μg / h. In some embodiments, an average fetuin-A fragment excretion rate greater than about 0.8 μg / h can be greater than about 2.3 μg / h. In some embodiments, the average fetuin-A fragment excretion rate greater than about 0.8 μg / h may be greater than about 2.5 μg / h.

[0126] In some embodiments, the hazard ratio corresponding to an average fetuin-A fragment excretion rate of about 0.9 μg / h to about 2.4 μg / h and an average fetuin-A fragment excretion rate of about 0.9 μg / h or less may be about 2 or greater. In some embodiments, the hazard ratio corresponding to an average fetuin-A fragment excretion rate of about 0.9 μg / h to about 2.4 μg / h and an average fetuin-A fragment excretion rate of about 0.9 μg / h or less may be about 1.5 or greater. In some embodiments, the hazard ratio corresponding to an average fetuin-A fragment excretion rate of about 0.9 μg / h to about 2.4 μg / h and an average fetuin-A fragment excretion rate of about 0.9 μg / h or less may be about 1.59 or greater. In some embodiments, the hazard ratio corresponding to an average fetuin-A fragment excretion rate of about 0.9 μg / h to about 2.4 μg / h and an average fetuin-A fragment excretion rate of about 0.9 μg / h or less may be about 1.6 or greater. In some embodiments, the hazard ratio corresponding to an average fetuin-A fragment excretion rate of about 0.9 μg / h to about 2.4 μg / h and an average fetuin-A fragment excretion rate of about 0.9 μg / h or less may be about 1.7 or greater. In some embodiments, the hazard ratio corresponding to an average fetuin-A fragment excretion rate of about 0.9 μg / h to about 2.4 μg / h and an average fetuin-A fragment excretion rate of about 0.9 μg / h or less may be about 1.78 or greater. In some embodiments, the hazard ratio corresponding to an average fetuin-A fragment excretion rate of about 0.9 μg / h to about 2.4 μg / h and an average fetuin-A fragment excretion rate of about 0.9 μg / h or less may be about 1.79 or greater. In some embodiments, the hazard ratio corresponding to an average fetuin-A fragment excretion rate of about 0.9 μg / h to about 2.4 μg / h and an average fetuin-A fragment excretion rate of about 0.9 μg / h or less may be about 1.8 or greater.In some embodiments, the hazard ratio corresponding to an average fetuin-A fragment excretion rate of about 0.9 μg / h to about 2.4 μg / h and an average fetuin-A fragment excretion rate of about 0.9 μg / h or less may be about 2.1 or greater. In some embodiments, the hazard ratio corresponding to an average fetuin-A fragment excretion rate of about 0.9 μg / h to about 2.4 μg / h and an average fetuin-A fragment excretion rate of about 0.9 μg / h or less may be about 2.15 or greater. In some embodiments, the hazard ratio corresponding to an average fetuin-A fragment excretion rate of about 0.9 μg / h to about 2.4 μg / h and an average fetuin-A fragment excretion rate of about 0.9 μg / h or less may be about 2.18 or greater. In some embodiments, the hazard ratio corresponding to an average fetuin-A fragment excretion rate of about 0.9 μg / h to about 2.4 μg / h and an average fetuin-A fragment excretion rate of about 0.9 μg / h or less may be about 2.2 or greater. In some embodiments, the hazard ratio corresponding to an average fetuin-A fragment excretion rate of about 0.9 μg / h to about 2.4 μg / h and an average fetuin-A fragment excretion rate of about 0.9 μg / h or less may be about 2.22 or greater.

[0127] In some embodiments, the hazard ratio corresponding to an average fetuin-A fragment excretion rate of about 0.9 μg / h to about 2.4 μg / h and an average fetuin-A fragment excretion rate of about 0.9 μg / h or less may be about 1.5 to about 2.5. In some embodiments, the hazard ratio corresponding to an average fetuin-A fragment excretion rate of about 0.9 μg / h to about 2.4 μg / h and an average fetuin-A fragment excretion rate of about 0.9 μg / h or less may be about 1.6 to about 2.3. In some embodiments, the hazard ratio corresponding to an average fetuin-A fragment excretion rate of about 0.9 μg / h to about 2.4 μg / h and an average fetuin-A fragment excretion rate of about 0.9 μg / h or less may be about 1.59 to about 2.22.

[0128] In some embodiments, the hazard ratio corresponding to an average fetuin-A fragment excretion rate of about 2.4 μg / h or more and an average fetuin-A fragment excretion rate of about 0.9 μg / h or less may be about 2 or greater. In some embodiments, the hazard ratio corresponding to an average fetuin-A fragment excretion rate of about 2.4 μg / h or more and an average fetuin-A fragment excretion rate of about 0.9 μg / h or less may be about 2.2 or greater. In some embodiments, the hazard ratio corresponding to an average fetuin-A fragment excretion rate of about 2.4 μg / h or more and an average fetuin-A fragment excretion rate of about 0.9 μg / h or less may be about 2.26 or greater. In some embodiments, the hazard ratio corresponding to an average fetuin-A fragment excretion rate of about 2.4 μg / h or more and an average fetuin-A fragment excretion rate of about 0.9 μg / h or less may be about 2.5 or greater. In some embodiments, the hazard ratio corresponding to an average fetuin-A fragment excretion rate of about 2.4 μg / h or more and an average fetuin-A fragment excretion rate of about 0.9 μg / h or less may be about 2.7 or greater. In some embodiments, the hazard ratio corresponding to an average fetuin-A fragment excretion rate of about 2.4 μg / h or more and an average fetuin-A fragment excretion rate of about 0.9 μg / h or less may be about 3 or greater. In some embodiments, the hazard ratio corresponding to an average fetuin-A fragment excretion rate of about 2.4 μg / h or more and an average fetuin-A fragment excretion rate of about 0.9 μg / h or less may be about 3.5 or more, hi some embodiments, the hazard ratio corresponding to an average fetuin-A fragment excretion rate of about 2.4 μg / h or more and an average fetuin-A fragment excretion rate of about 0.9 μg / h or less may be about 3.6 or more.

[0129] In some embodiments, the hazard ratio corresponding to an average fetuin-A fragment excretion rate of about 2.4 μg / h or more and an average fetuin-A fragment excretion rate of about 0.9 μg / h or less may be about 3.7 or greater. In some embodiments, the hazard ratio corresponding to an average fetuin-A fragment excretion rate of about 2.4 μg / h or more and an average fetuin-A fragment excretion rate of about 0.9 μg / h or less may be about 3.8 or greater. In some embodiments, the hazard ratio corresponding to an average fetuin-A fragment excretion rate of about 2.4 μg / h or more and an average fetuin-A fragment excretion rate of about 0.9 μg / h or less may be about 4 or greater. In some embodiments, the hazard ratio corresponding to an average fetuin-A fragment excretion rate of about 2.4 μg / h or more and an average fetuin-A fragment excretion rate of about 0.9 μg / h or less may be about 4.5 or greater. In some embodiments, the hazard ratio corresponding to an average fetuin-A fragment excretion rate of about 2.4 μg / h or more and an average fetuin-A fragment excretion rate of about 0.9 μg / h or less may be about 4.7 or greater. In some embodiments, the hazard ratio corresponding to an average fetuin-A fragment excretion rate of about 2.4 μg / h or more and an average fetuin-A fragment excretion rate of about 0.9 μg / h or less may be about 4.8 or greater. In some embodiments, the hazard ratio corresponding to an average fetuin-A fragment excretion rate of about 2.4 μg / h or more and an average fetuin-A fragment excretion rate of about 0.9 μg / h or less may be about 4.9 or greater. In some embodiments, the hazard ratio corresponding to an average fetuin-A fragment excretion rate of about 2.4 μg / h or more and an average fetuin-A fragment excretion rate of about 0.9 μg / h or less may be about 5 or greater.

[0130] In some embodiments, the hazard ratio corresponding to an average fetuin A fragment excretion rate of about 2.4 μg / h or more and an average fetuin A fragment excretion rate of about 0.9 μg / h or less may be about 2 to about 5.

[0131] In some embodiments, the hazard ratio corresponding to an average fetuin A fragment excretion rate of about 2.4 μg / h or more and an average fetuin A fragment excretion rate of about 0.9 μg / h or less may be about 2 to about 4.5.

[0132] In some embodiments, the hazard ratio corresponding to an average fetuin A fragment excretion rate of about 2.4 μg / h or more and an average fetuin A fragment excretion rate of about 0.9 μg / h or less may be about 2.5 to about 4.5.

[0133] In some embodiments, interacting with the fetuin-A fragment comprises binding to fetuin-A.

[0134] In some embodiments, the reagent comprises an antibody.

[0135] uC-FetA excretion correlates with renal function and is also associated with increased markers of proximal tubular injury and 24-hour urinary protein excretion. Importantly, uC-FetA excretion is independently associated with graft decline in stable KTRs, especially in patients with relatively good renal function.

[0136] In some embodiments, in KTRs, shorter time since transplantation, surviving donors, older donor age, use of growth inhibitors, and lower AST levels may be independently associated with increased 24-hour uC-FetA excretion. In some embodiments, 24-hour urinary L-FABP excretion, a marker of proximal tubule injury, may also be independently associated with increased 24-hour uC-FetA excretion. In some embodiments, increased uC-FetA excretion may be associated with an increased risk of graft dysfunction, even after adjusting for potential confounding factors. In healthy adults, the kidney does not express fetuin A. However, upon injury, proximal tubule epithelial cells (PTECs) can express and release fetuin A to the luminal side of the tubule. In cisplatin-induced and ischemia / reperfusion-induced AKI rat models, urinary fetuin A can be predominantly present in the urinary exosome fraction, rather than from the non-exosome fraction. PTECs can locally produce fetuin A under hypoxic conditions after stimulation from hypoxia-inducible transcription factors. The presence of fetuin A within the proximal tubule may help protect the kidney from hypoxia-induced renal inflammation by preventing the shift of macrophages to the proinflammatory M1 macrophage and from hypoxia-induced fibrosis by antagonizing TGFβ signaling.

[0137] The human precursor protein of fetuin A consists of three parts: the A chain, the connecting peptide, and the B chain, which are 321, 40, and 27 amino acids long, respectively. The connecting peptide of the precursor is removed by post-translational modification (i.e., limited proteolysis), after which only the A and B chains form the active fetuin A protein. The monoclonal antibody in the ELISA kit can detect fetuin A containing the connecting peptide. Therefore, the uC-FetA detected by this ELISA kit may be inactive fetuin A without protective effects on renal function, and elevated uC-FetA has been associated with the risk of reduced eGFR in patients. In addition to local production, fetuin A in urine can also originate from the circulation. Fetuin A is a negatively charged molecule with a molecular weight of approximately 60 kilodaltons, similar to that of albumin. Under physiological conditions, a selective amount of albumin can cross the glomerular filtration barrier (GFB), where it is reabsorbed by proximal tubular epithelial cells (PTEC) within the tubular compartment. Because fetuin-A and albumin have similar properties, comparable processing by the kidney can be expected. Fetuin-A can be absorbed into the proximal tubule of healthy rats by megalin-mediated endocytosis, primarily in the S1 segment and, to a lesser extent, in the S2 segment. Under conditions of accumulated glomerular damage, the GFB is disrupted, resulting in excessive leakage of different plasma proteins into the tubular compartment. This increases the amount of filtered proteins that the proximal tubule must reabsorb. Because the reabsorption capacity of PTEC is not infinite, saturation of this mechanism leads to the presence of proteins from the circulation in the urine.The filtered proteins can then cause damage within the tubules through various mechanisms: by clogging the tubular lumen with protein casts, by causing energy depletion and lysosomal rupture due to the excessive protein overload that PTECs must reabsorb, by triggering PTECs to release inflammatory and profibrotic cytokines, by causing the intracellular accumulation of lipidated proteins that induce tubular apoptosis, and by activating the complement system, causing direct damage to PTECs. When this occurs, the ability of PTECs to locally produce fetuin A and reabsorb filtered fetuin A may be impaired. Because 24-hour uC-FetA excretion was significantly associated with an increased risk of graft failure, even after adjusting for potential confounding factors, including 24-hour urinary protein excretion, the origin of uC-FetA may be due to local production by PTECs rather than an impaired reabsorption process. In sensitivity analyses, uC-FetA may not be associated with decreased graft function in patients with eGFR <30 ml / min / 1.73 m2. Fetuin A from the circulation may also end up in the urine if there is accumulated kidney damage. Therefore, uC-FetA measured in this patient subgroup may consist of locally produced fetuin A and circulating fetuin A. Circulating fetuin A is not associated with renal function.

[0138] Both acute and chronic kidney injury are closely associated with the development of hypoxia. Among all structures within the kidney, PTECs are the most vulnerable due to their high activity and oxygen demand. Because hypoxia plays a major role in the progression of kidney disease and can be present relatively early in kidney injury, even before the onset of structural damage, early identification of this condition may be beneficial to prevent further deterioration. In both cisplatin-induced and ischemia / reperfusion-induced AKI, urinary fetuin-A excretion increased before the rapid rise in serum creatinine. Furthermore, fetuin-A deposition in PTECs increased before morphologically present structural damage to the renal tubules. In type 2 diabetes patients, urinary fetuin-A may indicate the progression of renal function decline earlier than albuminuria. Since one of the most important requirements for a biomarker is that it reflects the underlying pathophysiology of the disease, in some embodiments, measurement of urinary fetuin A may offer additional advantages as a biomarker for early detection of graft damage in KTR over currently used parameters such as serum creatinine or proteinuria.

[0139] Assay In some embodiments, various methods, reagents, devices, and kits can be used to measure levels of fetuin A or fetuin A-based fragments in various media, such as urine, serum, and other bodily fluids. In some embodiments, enzyme-linked immunosorbent assays (ELISAs) can be used to measure fetuin A levels. For example, ELISAs can be used to quantitatively measure fetuin A with specific post-translational modifications (PTMs) in human urine and should be performed in a competent clinical laboratory by a certified medical professional, such as a medical technician. For example, a calibrator or unknown urine sample is mixed with an antibody that has binding affinity for a portion of fetuin A, such as a monoclonal antibody (mAb) against fetuin A with a specific PTM, and then incubated in a microplate pre-bound with fetuin A or a segment thereof, such as fetuin A with a specific PTM. The monoclonal antibody recognizes the fetuin A in the calibrator or unknown sample in competition with the antibody in the microplate well. After incubation, in some embodiments, a signal-generating molecule, such as a signal molecule-tagged antibody, can be used. In some embodiments, a horseradish peroxide (HRP)-conjugated secondary antibody is added, followed by incubation with 3,3',5,5'-tetramethylbenzidine (TMB) substrate. Their relative reactivities are determined by absorbance measurements at 450 nanometers (nm) and plotted against a predetermined specific PTM fetuin A calibration curve.

[0140] First Set of Non-Limiting Embodiments The present disclosure is also described by the following first set of non-limiting embodiments. However, the use of these and other embodiments anywhere in the specification is for illustrative purposes only and in no way limits the scope and meaning of the present disclosure. Likewise, the present disclosure is not limited to any particular preferred embodiment or aspect described herein. Indeed, modifications and variations may be apparent to those skilled in the art upon reading this specification, and such variations can be made without departing from the spirit or scope of the present disclosure.

[0141] 1. A method of preparing an assay, comprising: providing a first solution comprising a urine sample from the kidney transplant recipient after kidney transplantation and a first reagent for interacting with post-translationally modified fetuin in the urine fragment to determine the level of the fetuin A fragment in the urine sample; providing a second solution comprising the urine sample and a second reagent for interacting with urinary creatinine to determine the level of urinary creatinine in the urine sample; Correlating a ratio of the determined level of fetuin-A fragment to the determined level of urinary creatinine with a likelihood of graft failure in the recipient in response to the determined level of fetuin-A fragment and the determined level of urinary creatinine; The first solution and the second solution are the same solution or different solutions from each other; said correlating with one another; The method comprising:

[0142] 2. The method of embodiment 1, wherein the ratio greater than about 20 ng / mg, greater than about 21 ng / mg, greater than about 22 ng / mg, greater than about 23 ng / mg, more preferably greater than about 23.5 ng / mg, more preferably greater than about 24 ng / mg, triggers medical intervention to forestall said graft loss.

[0143] 3. The method of any one of the preceding embodiments, wherein the higher likelihood is higher than the likelihood of graft function decline over a period of about 10 years or less in a comparison recipient of a kidney transplant having the determined ratio of the level of fetuin-A fragment to the level of urinary creatinine of less than about 16 ng / mg, less than about 15.5 ng / mg, less than about 15 ng / mg, less than about 14.5 ng / mg, or less than about 14 ng / mg.

[0144] 4. The method of any one of the preceding embodiments, wherein said graft decline is indicated by a doubling of serum creatinine, the need for kidney retransplantation, or the need for dialysis.

[0145] 5. The method of any one of the preceding embodiments, wherein said graft function decline is indicated by a decline in estimated glomerular filtration rate (eGFR).

[0146] 6. The method of any one of the preceding embodiments, wherein said interacting with said fetuin A fragment comprises binding to said fetuin A fragment.

[0147] 7. The method of any one of the preceding embodiments, wherein said interacting with said urinary creatinine comprises binding to said urinary creatinine.

[0148] 8. The method of any one of the preceding embodiments, wherein at least one of the first reagent and the second reagent comprises an antibody.

[0149] 9. The method of any one of the preceding embodiments, wherein the ratio greater than about 20 ng / mg is greater than about 25 ng / mg.

[0150] 10. The method of any one of the preceding embodiments, wherein the ratio greater than about 20 ng / mg is greater than about 25.5 ng / mg.

[0151] 11. The method of any one of the preceding embodiments, wherein the ratio greater than about 20 ng / mg is greater than about 26 ng / mg.

[0152] 12. The method of any one of the preceding embodiments, wherein the ratio greater than about 20 ng / mg is greater than about 27 ng / mg.

[0153] 13. The method of any one of the preceding embodiments, wherein the ratio greater than about 20 ng / mg is greater than about 27.5 ng / mg.

[0154] 14. The method of any one of the preceding embodiments, wherein the ratio greater than about 20 ng / mg is greater than about 28 ng / mg.

[0155] 15. The method of any one of the preceding embodiments, wherein the ratio greater than about 20 ng / mg is greater than about 28.5 ng / mg.

[0156] 16. The method of any one of the preceding embodiments, wherein the ratio greater than about 20 ng / mg is greater than about 28.7 ng / mg.

[0157] 17. The method of any one of the preceding embodiments, wherein the ratio greater than about 20 ng / mg is greater than about 28.8 ng / mg.

[0158] 18. The method of any one of the preceding embodiments, wherein the ratio greater than about 20 ng / mg is greater than about 28.9 ng / mg.

[0159] 19. The method of any one of the preceding embodiments, wherein the ratio greater than about 20 ng / mg is greater than about 29.0 ng / mg.

[0160] 20. The method of any one of the preceding embodiments, wherein the ratio greater than about 20 ng / mg is greater than about 29.1 ng / mg.

[0161] 21. The method of any one of the preceding embodiments, wherein the ratio greater than about 20 ng / mg is greater than about 29.2 ng / mg.

[0162] 22. The method of any one of the preceding embodiments, wherein the ratio greater than about 20 ng / mg is greater than about 29.3 ng / mg.

[0163] 23. The method of any one of the preceding embodiments, wherein the ratio greater than about 20 ng / mg is greater than about 30 ng / mg.

[0164] 24. The method of any one of the preceding embodiments, wherein the ratio greater than about 20 ng / mg is greater than about 34 ng / mg.

[0165] 25. The method of any one of the preceding embodiments, wherein the ratio greater than about 20 ng / mg is greater than about 35 ng / mg.

[0166] 26. The method of any one of the preceding embodiments, wherein the ratio greater than about 20 ng / mg is greater than about 37 ng / mg.

[0167] 27. The method of any one of the preceding embodiments, wherein the ratio greater than about 20 ng / mg is greater than about 38 ng / mg.

[0168] 28. The method of any one of the preceding embodiments, wherein the ratio greater than about 20 ng / mg is greater than about 40 ng / mg.

[0169] 29. The method of any one of the preceding embodiments, wherein the ratio greater than about 20 ng / mg is greater than about 50 ng / mg.

[0170] 30. The method of any one of the preceding embodiments, wherein the ratio greater than about 20 ng / mg is greater than about 60 ng / mg.

[0171] 31. The method of any one of the preceding embodiments, wherein the ratio greater than about 20 ng / mg is greater than about 65 ng / mg.

[0172] 32. The method of any one of the preceding embodiments, wherein the ratio greater than about 20 ng / mg is greater than about 70 ng / mg.

[0173] 33. The method of any one of the preceding embodiments, wherein the ratio greater than about 20 ng / mg is greater than about 71 ng / mg.

[0174] 34. The method of any one of the preceding embodiments, wherein the ratio greater than about 20 ng / mg is greater than about 72 ng / mg.

[0175] 35. The method of any one of the preceding embodiments, wherein the ratio greater than about 20 ng / mg is greater than about 73 ng / mg.

[0176] 36. The method of any one of the preceding embodiments, wherein the ratio greater than about 20 ng / mg is greater than about 75 ng / mg.

[0177] 37. The method of any one of the preceding embodiments, wherein the ratio greater than about 20 ng / mg is greater than about 80 ng / mg.

[0178] 38. The method of any one of the preceding embodiments, wherein the ratio greater than about 20 ng / mg is greater than about 85 ng / mg.

[0179] 39. The method of any one of the preceding embodiments, wherein the ratio greater than about 20 ng / mg is greater than about 90 ng / mg.

[0180] 40. The method of any one of the preceding embodiments, wherein the ratio greater than about 20 ng / mg is greater than about 91 ng / mg.

[0181] 41. The method of any one of the preceding embodiments, wherein the ratio greater than about 20 ng / mg is greater than about 92 ng / mg.

[0182] 42. The method of any one of the preceding embodiments, wherein the ratio greater than about 20 ng / mg is greater than about 93 ng / mg.

[0183] 43. The method of any one of the preceding embodiments, wherein the ratio greater than about 20 ng / mg is greater than about 94 ng / mg.

[0184] 44. The method of any one of the preceding embodiments, wherein the ratio greater than about 20 ng / mg is greater than about 90 ng / mg.

[0185] 45. The method of any one of the preceding embodiments, wherein the ratio greater than about 20 ng / mg is greater than about 95 ng / mg.

[0186] 46. ​​The method of any one of the preceding embodiments, wherein the second ratio, less than about 14 ng / mg, is less than about 13 ng / mg.

[0187] 47. The method of any one of the preceding embodiments, wherein the second ratio, less than about 14 ng / mg, is less than about 12 ng / mg.

[0188] 48. The method of any one of the preceding embodiments, wherein the second ratio, less than about 14 ng / mg, is less than about 11 ng / mg.

[0189] 49. The method of any one of the preceding embodiments, wherein the second ratio, which is less than about 14 ng / mg, is less than about 10 ng / mg.

[0190] 50. The method of any one of the preceding embodiments, wherein the second ratio, less than about 14 ng / mg, is less than about 9 ng / mg.

[0191] 51. The method of any one of the preceding embodiments, wherein the second ratio less than about 14 ng / mg is less than about 8.8 ng / mg.

[0192] 52. The method of any one of the preceding embodiments, wherein the second ratio less than about 14 ng / mg is less than about 8.5 ng / mg.

[0193] 53. The method of any one of the preceding embodiments, wherein the second ratio less than about 14 ng / mg is less than about 8.4 ng / mg.

[0194] 54. The method of any one of the preceding embodiments, wherein the second ratio less than about 14 ng / mg is less than about 8.2 ng / mg.

[0195] 55. The method of any one of the preceding embodiments, wherein the second ratio less than about 14 ng / mg is less than about 8 ng / mg.

[0196] 56. The method of any one of the preceding embodiments, wherein the second ratio less than about 14 ng / mg is less than about 7.5 ng / mg.

[0197] 57. The method of any one of the preceding embodiments, wherein the second ratio less than about 14 ng / mg is less than about 7.4 ng / mg.

[0198] 58. The method of any one of the preceding embodiments, wherein the second ratio less than about 14 ng / mg is less than about 3.3 ng / mg.

[0199] 59. The method of any one of the preceding embodiments, wherein the second ratio greater than about 14 ng / mg is less than about 7.3 ng / mg.

[0200] 60. The method of any one of the preceding embodiments, wherein the second ratio less than about 14 ng / mg is less than about 7.2 ng / mg.

[0201] 61. The method of any one of the preceding embodiments, wherein the second ratio less than about 14 ng / mg is less than about 7.1 ng / mg.

[0202] 62. The method of any one of the preceding embodiments, wherein the second ratio, less than about 14 ng / mg, is less than about 7 ng / mg.

[0203] 63. The method of any one of the preceding embodiments, wherein the second ratio, less than about 14 ng / mg, is less than about 6 ng / mg.

[0204] 64. The method of any one of the preceding embodiments, wherein the second ratio greater than about 14 ng / mg is less than about 5 ng / mg.

[0205] 65. The method of any one of the preceding embodiments, wherein the second ratio, less than about 14 ng / mg, is less than about 4 ng / mg.

[0206] 66. The method of any one of the preceding embodiments, wherein the second ratio less than about 14 ng / mg is less than about 3.9 ng / mg.

[0207] 67. The method of any one of the preceding embodiments, wherein the second ratio less than about 14 ng / mg is less than about 3.8 ng / mg.

[0208] 68. The method of any one of the preceding embodiments, wherein the second ratio less than about 14 ng / mg is less than about 3.7 ng / mg.

[0209] 69. The method of any one of the preceding embodiments, wherein the second ratio less than about 14 ng / mg is less than about 3.6 ng / mg.

[0210] 70. The method of any one of the preceding embodiments, wherein the second ratio less than about 14 ng / mg is less than about 3.5 ng / mg.

[0211] 71. The method of any one of the preceding embodiments, wherein the higher likelihood of graft failure is greater than or equal to about 5%.

[0212] 72. The method of any one of the preceding embodiments, wherein the higher likelihood of graft failure is greater than or equal to about 8%.

[0213] 73. The method of any one of the preceding embodiments, wherein the higher likelihood of graft failure is greater than or equal to about 10%.

[0214] 74. The method of any one of the preceding embodiments, wherein the higher likelihood of graft failure is greater than or equal to about 15%.

[0215] 75. The method of any one of the preceding embodiments, wherein the higher likelihood of graft failure is greater than or equal to about 16%.

[0216] 76. The method of any one of the preceding embodiments, wherein the higher likelihood of graft failure is greater than or equal to about 20%.

[0217] 77. The method of any one of the preceding embodiments, wherein the higher likelihood of graft failure is greater than or equal to about 22%.

[0218] 78. The method of any one of the preceding embodiments, wherein the higher likelihood of graft failure is greater than or equal to about 25%.

[0219] 79. The method of any one of the preceding embodiments, wherein the higher likelihood of graft failure is greater than or equal to about 30%.

[0220] 80. The method of any one of the preceding embodiments, wherein the higher likelihood of graft failure is greater than or equal to about 32%.

[0221] 81. The method of any one of the preceding embodiments, wherein the higher likelihood of graft failure is greater than or equal to about 35%.

[0222] 82. The method of any one of the preceding embodiments, wherein the higher likelihood of graft failure is greater than or equal to about 36%.

[0223] 83. The method of any one of the preceding embodiments, wherein the higher likelihood of graft failure is greater than or equal to about 40%.

[0224] 84. The method of any one of the preceding embodiments, wherein the higher likelihood of graft failure is greater than or equal to about 45%.

[0225] 85. The method of any one of the preceding embodiments, wherein the higher likelihood of graft failure is greater than or equal to about 50%.

[0226] 86. The method of any one of the preceding embodiments, wherein the higher likelihood of graft dysfunction is between about 5% and about 50%.

[0227] 87. The method of any one of the preceding embodiments, wherein the higher likelihood of graft dysfunction is between about 10% and about 45%.

[0228] 88. The method of any one of the preceding embodiments, wherein the higher likelihood of graft dysfunction is between about 20% and about 40%.

[0229] 89. The method of any one of the preceding embodiments, wherein the higher likelihood of graft dysfunction is between about 25% and about 35%.

[0230] 90. The method of any one of the preceding embodiments, wherein the higher likelihood of graft dysfunction is about 25% to about 30%.

[0231] 91. The method of any one of the preceding embodiments, wherein the higher likelihood of graft dysfunction is between about 10% and about 20%.

[0232] 92. The method of any one of the preceding embodiments, wherein the higher likelihood of graft dysfunction is between about 15% and about 25%.

[0233] 93. The method of any one of the preceding embodiments, wherein the higher likelihood of graft dysfunction is between about 20% and about 30%.

[0234] 94. The method of any one of the preceding embodiments, wherein the higher likelihood of graft dysfunction is between about 30% and about 40%.

[0235] 95. The method of any one of the preceding embodiments, wherein the higher likelihood of graft dysfunction is between about 35% and about 45%.

[0236] 96. The method of any one of the preceding embodiments, wherein the higher likelihood of graft dysfunction is between about 40% and about 50%.

[0237] 97. The method of any one of the preceding embodiments, wherein the graft failure occurs after two years.

[0238] 98. The method of any one of the preceding embodiments, wherein the graft failure occurs after 3 years.

[0239] 99. The method of any one of the preceding embodiments, wherein the graft failure occurs after 4 years.

[0240] 100. The method of any one of the preceding embodiments, wherein the graft failure occurs after 5 years.

[0241] 101. The method of any one of the preceding embodiments, wherein the graft failure occurs after 6 years.

[0242] 102. The method of any one of the preceding embodiments, wherein the graft failure occurs after 7 years.

[0243] 103. The method of any one of the preceding embodiments, wherein the graft failure occurs after 8 years.

[0244] 104. The method of any one of the preceding embodiments, wherein the graft failure occurs after 9 years.

[0245] 105. The method of any one of the preceding embodiments, wherein the graft failure occurs within 7 years.

[0246] 106. The method of any one of the preceding embodiments, wherein the graft failure occurs within 8 years.

[0247] 107. The method of any one of the preceding embodiments, wherein the graft failure occurs within 9 years.

[0248] 108. The method of any one of the preceding embodiments, further comprising assessing the probability of graft failure based on the ratio and at least one marker.

[0249] 109. The method of embodiment 108, wherein the at least one other marker comprises age, sex, time since transplant at enrollment, urinary albumin-to-creatine ratio (UACR), estimated glomerular filtration rate (eGFR) based on the creatinine and cystatin C-based CKD-EPI equation, the presence of human leukocyte antigen, or any combination thereof.

[0250] 110. The method of embodiment 109, wherein the assessed probability exhibits an area under the receiver operating characteristic (ROC) curve (AUC) of at least about 70 percent (%).

[0251] 111. The method of embodiment 110, wherein the assessed probability represents at least about 80 percent (%) of the AUC.

[0252] 112. The method of embodiment 110, wherein the assessed probability represents at least about 85 percent (%) of the AUC.

[0253] 113. The method of embodiment 110, wherein the assessed probability represents at least about 90 percent (%) of the AUC.

[0254] 114. The method of embodiment 110, wherein the assessed probability represents at least about 95 percent (%) of the AUC.

[0255] 115. An assay kit for determining the possibility of progressive renal function decline, comprising: a first solution comprising a first reagent for interacting with post-translationally modified fetuin-A fragments in urine fragments to indicate the level of the fetuin-A fragments in a urine sample from the kidney transplant recipient after kidney transplantation; a second solution comprising a second reagent for interacting with urinary creatinine to indicate the level of urinary creatinine in the urine sample; a device for determining the level of the fetuin A fragment and the level of the urinary creatinine in the urine sample and determining a ratio of the determined level of the fetuin A fragment to the determined level of the urinary creatinine, a ratio greater than about 20 ng / mg, greater than about 21 ng / mg, greater than about 22 ng / mg, greater than about 23 ng / mg, more preferably greater than about 23.5 ng / mg, more preferably greater than about 24 ng / mg indicates a greater likelihood of graft failure over a period of about 10 years or less; The first solution and the second solution are the same solution or different solutions from each other; the device; The assay kit comprising:

[0256] 116. The assay kit of embodiment 115, wherein a ratio greater than about 20 ng / mg, greater than about 21 ng / mg, greater than about 22 ng / mg, greater than about 23 ng / mg, more preferably greater than about 23.5 ng / mg, more preferably greater than about 24 ng / mg, triggers medical intervention to forestall said graft dysfunction.

[0257] 117. The assay kit of any one of the preceding embodiments after 115, wherein the higher likelihood is higher than the likelihood of graft function decline over a period of about 10 years or less in a comparison recipient of a kidney transplant having a lower ratio of the determined level of the fetuin-A fragment to the level of the urinary creatinine of less than about 16 ng / mg, less than about 15.5 ng / mg, less than about 15 ng / mg, less than about 14.5 ng / mg, or less than about 14 ng / mg.

[0258] 118. The assay kit of any one of the preceding embodiments after 115, wherein said graft decline is indicated by a doubling of serum creatinine, a need for kidney retransplantation, or a need for dialysis.

[0259] 119. The assay kit of any one of the preceding embodiments after 115, wherein said graft function decline is indicated by a decline in estimated glomerular filtration rate (eGFR).

[0260] 120. The assay kit of any one of the preceding embodiments after 115, wherein said interacting with said fetuin A fragment comprises binding to said fetuin A fragment.

[0261] 121. The assay kit of any one of the preceding embodiments after 115, wherein said interacting with said urinary creatinine comprises binding to said urinary creatinine.

[0262] 122. The assay kit of any one of the preceding embodiments after 115, wherein at least one of the first reagent and the second reagent comprises an antibody.

[0263] 123. The assay kit of any one of the preceding embodiments after 115, wherein the ratio greater than about 20 ng / mg is greater than about 25 ng / mg.

[0264] 124. The assay kit of any one of the preceding embodiments after 115, wherein the ratio greater than about 20 ng / mg is greater than about 25.5 ng / mg.

[0265] 125. The assay kit of any one of the preceding embodiments after 115, wherein the ratio greater than about 20 ng / mg is greater than about 26 ng / mg.

[0266] 126. The assay kit of any one of the preceding embodiments after 115, wherein the ratio greater than about 20 ng / mg is greater than about 27 ng / mg.

[0267] 127. The assay kit of any one of the preceding embodiments after 115, wherein the ratio greater than about 20 ng / mg is greater than about 27.5 ng / mg.

[0268] 128. The assay kit of any one of the preceding embodiments after 115, wherein the ratio greater than about 20 ng / mg is greater than about 28 ng / mg.

[0269] 129. The assay kit of any one of the preceding embodiments after 115, wherein the ratio greater than about 20 ng / mg is greater than about 28.5 ng / mg.

[0270] 130. The assay kit of any one of the preceding embodiments after 115, wherein the ratio greater than about 20 ng / mg is greater than about 28.7 ng / mg.

[0271] 131. The assay kit of any one of the preceding embodiments after 115, wherein the ratio greater than about 20 ng / mg is greater than about 28.8 ng / mg.

[0272] 132. The assay kit of any one of the preceding embodiments after 115, wherein the ratio greater than about 20 ng / mg is greater than about 28.9 ng / mg.

[0273] 133. The assay kit of any one of the preceding embodiments after 115, wherein the ratio greater than about 20 ng / mg is greater than about 29.0 ng / mg.

[0274] 134. The assay kit of any one of the preceding embodiments after 115, wherein the ratio greater than about 20 ng / mg is greater than about 29.1 ng / mg.

[0275] 135. The assay kit of any one of the preceding embodiments after 115, wherein the ratio greater than about 20 ng / mg is greater than about 29.2 ng / mg.

[0276] 136. The assay kit of any one of the preceding embodiments after 115, wherein the ratio greater than about 20 ng / mg is greater than about 29.3 ng / mg.

[0277] 137. The assay kit of any one of the preceding embodiments after 115, wherein the ratio greater than about 20 ng / mg is greater than about 30 ng / mg.

[0278] 138. The assay kit of any one of the preceding embodiments after 115, wherein the ratio greater than about 20 ng / mg is greater than about 34 ng / mg.

[0279] 139. The assay kit of any one of the preceding embodiments after 115, wherein the ratio greater than about 20 ng / mg is greater than about 35 ng / mg.

[0280] 140. The assay kit of any one of the preceding embodiments after 115, wherein the ratio greater than about 20 ng / mg is greater than about 37 ng / mg.

[0281] 141. The assay kit of any one of the preceding embodiments after 115, wherein the ratio greater than about 20 ng / mg is greater than about 38 ng / mg.

[0282] 142. The assay kit of any one of the preceding embodiments after 115, wherein the ratio greater than about 20 ng / mg is greater than about 40 ng / mg.

[0283] 143. The assay kit of any one of the preceding embodiments after 115, wherein the ratio greater than about 20 ng / mg is greater than about 50 ng / mg.

[0284] 144. The assay kit of any one of the preceding embodiments after 115, wherein the ratio greater than about 20 ng / mg is greater than about 60 ng / mg.

[0285] 145. The assay kit of any one of the preceding embodiments after 115, wherein the ratio greater than about 20 ng / mg is greater than about 65 ng / mg.

[0286] 146. The assay kit of any one of the preceding embodiments after 115, wherein the ratio greater than about 20 ng / mg is greater than about 70 ng / mg.

[0287] 147. The assay kit of any one of the preceding embodiments after 115, wherein the ratio greater than about 20 ng / mg is greater than about 71 ng / mg.

[0288] 148. The assay kit of any one of the preceding embodiments after 115, wherein the ratio greater than about 20 ng / mg is greater than about 72 ng / mg.

[0289] 149. The assay kit of any one of the preceding embodiments after 115, wherein the ratio greater than about 20 ng / mg is greater than about 73 ng / mg.

[0290] 150. The assay kit of any one of the preceding embodiments after 115, wherein the ratio greater than about 20 ng / mg is greater than about 75 ng / mg.

[0291] 151. The assay kit of any one of the preceding embodiments after 115, wherein the ratio greater than about 20 ng / mg is greater than about 80 ng / mg.

[0292] 152. The assay kit of any one of the preceding embodiments after 115, wherein the ratio greater than about 20 ng / mg is greater than about 85 ng / mg.

[0293] 153. The assay kit of any one of the preceding embodiments after 115, wherein the ratio greater than about 20 ng / mg is greater than about 90 ng / mg.

[0294] 154. The assay kit of any one of the preceding embodiments after 115, wherein the ratio greater than about 20 ng / mg is greater than about 91 ng / mg.

[0295] 155. The assay kit of any one of the preceding embodiments after 115, wherein the ratio greater than about 20 ng / mg is greater than about 92 ng / mg.

[0296] 156. The assay kit of any one of the preceding embodiments after 115, wherein the ratio greater than about 20 ng / mg is greater than about 93 ng / mg.

[0297] 157. The assay kit of any one of the preceding embodiments after 115, wherein the ratio greater than about 20 ng / mg is greater than about 94 ng / mg.

[0298] 158. The assay kit of any one of the preceding embodiments after 115, wherein the ratio greater than about 20 ng / mg is greater than about 90 ng / mg.

[0299] 159. The assay kit of any one of the preceding embodiments after 115, wherein the ratio greater than about 20 ng / mg is greater than about 95 ng / mg.

[0300] 160. The assay kit of any one of the preceding embodiments after 115, wherein the second ratio less than about 14 ng / mg is less than about 13 ng / mg.

[0301] 161. The assay kit of any one of the preceding embodiments after 115, wherein the second ratio less than about 14 ng / mg is less than about 12 ng / mg.

[0302] 162. The assay kit of any one of the preceding embodiments after 115, wherein the second ratio less than about 14 ng / mg is less than about 11 ng / mg.

[0303] 163. The assay kit of any one of the preceding embodiments after 115, wherein the second ratio less than about 14 ng / mg is less than about 10 ng / mg.

[0304] 164. The assay kit of any one of the preceding embodiments after 115, wherein the second ratio less than about 14 ng / mg is less than about 9 ng / mg.

[0305] 165. The assay kit of any one of the preceding embodiments after 115, wherein the second ratio less than about 14 ng / mg is less than about 8.8 ng / mg.

[0306] 166. The assay kit of any one of the preceding embodiments after 115, wherein the second ratio less than about 14 ng / mg is less than about 8.5 ng / mg.

[0307] 167. The assay kit of any one of the preceding embodiments after 115, wherein the second ratio less than about 14 ng / mg is less than about 8.4 ng / mg.

[0308] 168. The assay kit of any one of the preceding embodiments after 115, wherein the second ratio less than about 14 ng / mg is less than about 8.2 ng / mg.

[0309] 169. The assay kit of any one of the preceding embodiments after 115, wherein the second ratio less than about 14 ng / mg is less than about 8 ng / mg.

[0310] 170. The assay kit of any one of the preceding embodiments after 115, wherein the second ratio less than about 14 ng / mg is less than about 7.5 ng / mg.

[0311] 171. The assay kit of any one of the preceding embodiments after 115, wherein the second ratio less than about 14 ng / mg is less than about 7.4 ng / mg.

[0312] 172. The assay kit of any one of the preceding embodiments after 115, wherein the second ratio less than about 14 ng / mg is less than about 3.3 ng / mg.

[0313] 173. The assay kit of any one of the preceding embodiments after 115, wherein the second ratio less than about 14 ng / mg is less than about 7.3 ng / mg.

[0314] 174. The assay kit of any one of the preceding embodiments after 115, wherein the second ratio less than about 14 ng / mg is less than about 7.2 ng / mg.

[0315] 175. The assay kit of any one of the preceding embodiments after 115, wherein the second ratio less than about 14 ng / mg is less than about 7.1 ng / mg.

[0316] 176. The assay kit of any one of the preceding embodiments after 115, wherein the second ratio less than about 14 ng / mg is less than about 7 ng / mg.

[0317] 177. The assay kit of any one of the preceding embodiments after 115, wherein the second ratio less than about 14 ng / mg is less than about 14 ng / mg.

[0318] 178. The assay kit of any one of the preceding embodiments after 115, wherein the second ratio less than about 14 ng / mg is less than about 5 ng / mg.

[0319] 179. The assay kit of any one of the preceding embodiments after 115, wherein the second ratio less than about 14 ng / mg is less than about 4 ng / mg.

[0320] 180. The assay kit of any one of the preceding embodiments after 115, wherein the second ratio less than about 14 ng / mg is less than about 3.9 ng / mg.

[0321] 181. The assay kit of any one of the preceding embodiments after 115, wherein the second ratio less than about 14 ng / mg is less than about 3.8 ng / mg.

[0322] 182. The assay kit of any one of the preceding embodiments after 115, wherein the second ratio less than about 14 ng / mg is less than about 3.7 ng / mg.

[0323] 183. The assay kit of any one of the preceding embodiments after 115, wherein the second ratio less than about 14 ng / mg is less than about 3.6 ng / mg.

[0324] 184. The assay kit of any one of the preceding embodiments after 115, wherein the second ratio less than about 14 ng / mg is less than about 3.5 ng / mg.

[0325] 185. The assay kit of any one of the preceding embodiments after 115, wherein the higher likelihood of graft dysfunction is about 5% or greater.

[0326] 186. The assay kit of any one of the preceding embodiments after 115, wherein the higher likelihood of graft failure is about 8% or greater.

[0327] 187. The assay kit of any one of the preceding embodiments after 115, wherein the higher likelihood of graft dysfunction is about 10% or greater.

[0328] 188. The assay kit of any one of the preceding embodiments after 115, wherein the higher likelihood of graft dysfunction is about 15% or greater.

[0329] 189. The assay kit of any one of the preceding embodiments after 115, wherein the higher likelihood of graft failure is about 16% or greater.

[0330] 190. The assay kit of any one of the preceding embodiments after 115, wherein the higher likelihood of graft failure is about 20% or greater.

[0331] 191. The assay kit of any one of the preceding embodiments after 115, wherein the higher likelihood of graft failure is about 22% or greater.

[0332] 192. The assay kit of any one of the preceding embodiments after 115, wherein the higher likelihood of graft failure is about 25% or greater.

[0333] 193. The assay kit of any one of the preceding embodiments after 115, wherein the higher likelihood of graft failure is about 30% or greater.

[0334] 194. The assay kit of any one of the preceding embodiments after 115, wherein the higher likelihood of graft failure is about 32% or greater.

[0335] 195. The assay kit of any one of the preceding embodiments after 115, wherein the higher likelihood of graft failure is about 35% or greater.

[0336] 196. The assay kit of any one of the preceding embodiments after 115, wherein the higher likelihood of graft failure is about 36% or greater.

[0337] 197. The assay kit of any one of the preceding embodiments after 115, wherein the higher likelihood of graft failure is about 40% or greater.

[0338] 198. The assay kit of any one of the preceding embodiments after 115, wherein the higher likelihood of graft failure is about 45% or greater.

[0339] 199. The assay kit of any one of the preceding embodiments after 115, wherein the higher likelihood of graft dysfunction is about 50% or greater.

[0340] 200. The assay kit of any one of the preceding embodiments after 115, wherein the higher likelihood of graft dysfunction is between about 5% and about 50%.

[0341] 201. The assay kit of any one of the preceding embodiments after 115, wherein the higher likelihood of graft dysfunction is between about 10% and about 40%.

[0342] 202. The assay kit of any one of the preceding embodiments after 115, wherein the higher likelihood of graft dysfunction is between about 20% and about 40%.

[0343] 203. The assay kit of any one of the preceding embodiments after 115, wherein the higher likelihood of graft dysfunction is between about 25% and about 35%.

[0344] 204. The assay kit of any one of the preceding embodiments after 115, wherein the higher likelihood of graft dysfunction is between about 25% and about 30%.

[0345] 205. The assay kit of any one of the preceding embodiments after 115, wherein the higher likelihood of graft dysfunction is between about 10% and about 20%.

[0346] 206. The assay kit of any one of the preceding embodiments after 115, wherein the higher likelihood of graft dysfunction is between about 15% and about 25%.

[0347] 207. The assay kit of any one of the preceding embodiments after 115, wherein the higher likelihood of graft dysfunction is between about 20% and about 30%.

[0348] 208. The assay kit of any one of the preceding embodiments after 115, wherein the higher likelihood of graft dysfunction is between about 30% and about 40%.

[0349] 209. The assay kit of any one of the preceding embodiments after 115, wherein the higher likelihood of graft dysfunction is between about 35% and about 45%.

[0350] 210. The assay kit of any one of the preceding embodiments after 115, wherein the higher likelihood of graft dysfunction is between about 40% and about 50%.

[0351] 211. The assay kit of any one of the preceding embodiments after 115, wherein the graft failure occurs after two years.

[0352] 212. The assay kit of any one of the preceding embodiments after 115, wherein the graft failure occurs after 3 years.

[0353] 213. The assay kit of any one of the preceding embodiments after 115, wherein the graft failure occurs after 4 years.

[0354] 214. The assay kit of any one of the preceding embodiments after 115, wherein the graft failure occurs after 5 years.

[0355] 215. The assay kit of any one of the preceding embodiments after 115, wherein said graft failure occurs after 6 years.

[0356] 216. The assay kit of any one of the preceding embodiments after 115, wherein said graft failure occurs after 7 years.

[0357] 217. The assay kit of any one of the preceding embodiments after 115, wherein the graft failure occurs after 8 years.

[0358] 218. The assay kit of any one of the preceding embodiments after 115, wherein the graft failure occurs after 9 years.

[0359] 219. The assay kit of any one of the preceding embodiments after 115, wherein the graft failure occurs within 7 years.

[0360] 220. The assay kit of any one of the preceding embodiments after 115, wherein the graft failure occurs within 8 years.

[0361] 221. The assay kit of any one of the preceding embodiments after 115, wherein the graft failure occurs within 9 years.

[0362] 222. The assay kit of any one of the preceding embodiments after 115, wherein the device is for determining the level of the fetuin A fragment and the level of the urinary creatinine to assess the probability of graft dysfunction based on the ratio and at least one other marker.

[0363] 223. The assay kit of embodiment 222, wherein the at least one other marker comprises age, sex, time since transplant at enrollment, urinary albumin-to-creatine ratio (UACR), estimated glomerular filtration rate (eGFR) based on the creatinine and cystatin C-based CKD-EPI equation, presence of human leukocyte antigen, or any combination thereof.

[0364] 224. The assay kit of embodiment 223, wherein the assessed probability exhibits an area under the receiver operating characteristic (ROC) curve (AUC) of at least about 70 percent (%).

[0365] 225. The assay kit of embodiment 224, wherein the assessed probability represents at least about 80 percent (%) of the AUC.

[0366] 226. The assay kit of embodiment 224, wherein the assessed probability represents at least about 85 percent (%) of the AUC.

[0367] 227. The assay kit of embodiment 224, wherein the assessed probability represents at least about 90 percent (%) of the AUC.

[0368] 228. The assay kit of embodiment 224, wherein the assessed probability represents at least about 95 percent (%) of the AUC.

[0369] 229. A method for correlating the likelihood of graft failure, comprising: determining levels of a first biomarker and a second biomarker in a urine sample from the kidney transplant recipient after kidney transplantation, wherein the first biomarker is a post-translationally modified fetuin A fragment in urine and the second biomarker is urinary creatinine; determining the ratio of the level of the first biomarker to the level of the second biomarker; correlating said ratio with a likelihood of graft failure in said recipient over a period of about 10 years; A ratio greater than about 20 ng / mg, greater than about 21 ng / mg, greater than about 22 ng / mg, greater than about 23 ng / mg, more preferably greater than about 23.5 ng / mg, more preferably greater than about 24 ng / mg indicates a greater likelihood of graft failure over the period of about 10 years or less. said correlating with one another; The method comprising:

[0370] 230. The method of embodiment 229, wherein the ratio is greater than about 20 ng / mg, greater than about 21 ng / mg, greater than about 22 ng / mg, greater than about 23 ng / mg, more preferably greater than about 23.5 ng / mg, and more preferably greater than about 24 ng / mg, triggering medical intervention to forestall said graft failure.

[0371] 231. The method of any one of the preceding embodiments after 229, wherein the higher likelihood is higher than the lower likelihood of graft decline over a period of about 10 years or less in a comparison recipient of a kidney transplant having the ratio of the determined level of fetuin-A fragment to the level of urinary creatinine of less than about 16 ng / mg, less than about 15.5 ng / mg, less than about 15 ng / mg, less than about 14.5 ng / mg, or less than about 14 ng / mg.

[0372] 232. The method of any one of the preceding embodiments after 229, wherein said graft decline is indicated by a doubling of serum creatinine, the need for kidney retransplantation, or the need for dialysis.

[0373] 233. The method of any one of the preceding embodiments after 229, wherein the graft function decline is indicated by a decline in estimated glomerular filtration rate (eGFR).

[0374] 234. The method of any one of the preceding embodiments after 229, wherein said interacting with said fetuin A fragment comprises binding to said fetuin A fragment.

[0375] 235. The method of any one of the preceding embodiments after 229, wherein said interacting with said urinary creatinine comprises binding to said urinary creatinine.

[0376] 236. The method of any one of the preceding embodiments after 229, wherein at least one of the first reagent and the second reagent comprises an antibody.

[0377] 237. The method of any one of the preceding embodiments after 229, wherein the ratio greater than about 20 ng / mg is greater than about 25 ng / mg.

[0378] 238. The method of any one of the preceding embodiments after 229, wherein the ratio greater than about 20 ng / mg is greater than about 25.5 ng / mg.

[0379] 239. The method of any one of the preceding embodiments after 229, wherein the ratio greater than about 20 ng / mg is greater than about 26 ng / mg.

[0380] 240. The method of any one of the preceding embodiments after 229, wherein the ratio greater than about 20 ng / mg is greater than about 27 ng / mg.

[0381] 241. The method of any one of the preceding embodiments after 229, wherein the ratio greater than about 20 ng / mg is greater than about 27.5 ng / mg.

[0382] 242. The method of any one of the preceding embodiments after 229, wherein the ratio greater than about 20 ng / mg is greater than about 28 ng / mg.

[0383] 243. The method of any one of the preceding embodiments after 229, wherein the ratio greater than about 20 ng / mg is greater than about 28.5 ng / mg.

[0384] 244. The method of any one of the preceding embodiments after 229, wherein the ratio greater than about 20 ng / mg is greater than about 28.7 ng / mg.

[0385] 245. The method of any one of the preceding embodiments after 229, wherein the ratio greater than about 20 ng / mg is greater than about 28.8 ng / mg.

[0386] 246. The method of any one of the preceding embodiments after 229, wherein the ratio greater than about 20 ng / mg is greater than about 28.9 ng / mg.

[0387] 247. The method of any one of the preceding embodiments after 229, wherein the ratio greater than about 20 ng / mg is greater than about 29.0 ng / mg.

[0388] 248. The method of any one of the preceding embodiments after 229, wherein the ratio greater than about 20 ng / mg is greater than about 29.1 ng / mg.

[0389] 249. The method of any one of the preceding embodiments after 229, wherein the ratio greater than about 20 ng / mg is greater than about 29.2 ng / mg.

[0390] 250. The method of any one of the preceding embodiments after 229, wherein the ratio greater than about 20 ng / mg is greater than about 29.3 ng / mg.

[0391] 251. The method of any one of the preceding embodiments after 229, wherein the ratio greater than about 20 ng / mg is greater than about 30 ng / mg.

[0392] 252. The method of any one of the preceding embodiments after 229, wherein the ratio greater than about 20 ng / mg is greater than about 34 ng / mg.

[0393] 253. The method of any one of the preceding embodiments after 229, wherein the ratio greater than about 20 ng / mg is greater than about 35 ng / mg.

[0394] 254. The method of any one of the preceding embodiments after 229, wherein the ratio greater than about 20 ng / mg is greater than about 37 ng / mg.

[0395] 255. The method of any one of the preceding embodiments after 229, wherein the ratio greater than about 20 ng / mg is greater than about 38 ng / mg.

[0396] 256. The method of any one of the preceding embodiments after 229, wherein the ratio greater than about 20 ng / mg is greater than about 40 ng / mg.

[0397] 257. The method of any one of the preceding embodiments after 229, wherein the ratio greater than about 20 ng / mg is greater than about 50 ng / mg.

[0398] 258. The method of any one of the preceding embodiments after 229, wherein the ratio greater than about 20 ng / mg is greater than about 60 ng / mg.

[0399] 259. The method of any one of the preceding embodiments after 229, wherein the ratio greater than about 20 ng / mg is greater than about 65 ng / mg.

[0400] 260. The method of any one of the preceding embodiments after 229, wherein the ratio greater than about 20 ng / mg is greater than about 70 ng / mg.

[0401] 261. The method of any one of the preceding embodiments after 229, wherein the ratio greater than about 20 ng / mg is greater than about 71 ng / mg.

[0402] 262. The method of any one of the preceding embodiments after 229, wherein the ratio greater than about 20 ng / mg is greater than about 72 ng / mg.

[0403] 263. The method of any one of the preceding embodiments after 229, wherein the ratio greater than about 20 ng / mg is greater than about 73 ng / mg.

[0404] 264. The method of any one of the preceding embodiments after 229, wherein the ratio greater than about 20 ng / mg is greater than about 75 ng / mg.

[0405] 265. The method of any one of the preceding embodiments after 229, wherein the ratio greater than about 20 ng / mg is greater than about 80 ng / mg.

[0406] 266. The method of any one of the preceding embodiments after 229, wherein the ratio greater than about 20 ng / mg is greater than about 85 ng / mg.

[0407] 267. The method of any one of the preceding embodiments after 229, wherein the ratio greater than about 20 ng / mg is greater than about 90 ng / mg.

[0408] 268. The method of any one of the preceding embodiments after 229, wherein the ratio greater than about 20 ng / mg is greater than about 91 ng / mg.

[0409] 269. The method of any one of the preceding embodiments after 229, wherein the ratio greater than about 20 ng / mg is greater than about 92 ng / mg.

[0410] 270. The method of any one of the preceding embodiments after 229, wherein the ratio greater than about 20 ng / mg is greater than about 93 ng / mg.

[0411] 271. The method of any one of the preceding embodiments after 229, wherein the ratio greater than about 20 ng / mg is greater than about 94 ng / mg.

[0412] 272. The method of any one of the preceding embodiments after 229, wherein the ratio greater than about 20 ng / mg is greater than about 90 ng / mg.

[0413] 273. The method of any one of the preceding embodiments after 229, wherein the ratio greater than about 20 ng / mg is greater than about 95 ng / mg.

[0414] 274. The method of any one of the preceding embodiments after 229, wherein the second ratio less than about 14 ng / mg is less than about 13 ng / mg.

[0415] 275. The method of any one of the preceding embodiments after 229, wherein the second ratio less than about 14 ng / mg is less than about 12 ng / mg.

[0416] 276. The method of any one of the preceding embodiments after 229, wherein the second ratio less than about 14 ng / mg is less than about 11 ng / mg.

[0417] 277. The method of any one of the preceding embodiments after 229, wherein the second ratio less than about 14 ng / mg is less than about 10 ng / mg.

[0418] 278. The method of any one of the preceding embodiments after 229, wherein the second ratio less than about 14 ng / mg is less than about 9 ng / mg.

[0419] 279. The method of any one of the preceding embodiments after 229, wherein the second ratio less than about 14 ng / mg is less than about 8.8 ng / mg.

[0420] 280. The method of any one of the preceding embodiments after 229, wherein the second ratio less than about 14 ng / mg is less than about 8.5 ng / mg.

[0421] 281. The method of any one of the preceding embodiments after 229, wherein the second ratio less than about 14 ng / mg is less than about 8.4 ng / mg.

[0422] 282. The method of any one of the preceding embodiments after 229, wherein the second ratio less than about 14 ng / mg is less than about 8.2 ng / mg.

[0423] 283. The method of any one of the preceding embodiments after 229, wherein the second ratio less than about 14 ng / mg is less than about 8 ng / mg.

[0424] 284. The method of any one of the preceding embodiments after 229, wherein the second ratio less than about 14 ng / mg is less than about 7.5 ng / mg.

[0425] 285. The method of any one of the preceding embodiments after 229, wherein the second ratio less than about 14 ng / mg is less than about 7.4 ng / mg.

[0426] 286. The method of any one of the preceding embodiments after 229, wherein the second ratio less than about 14 ng / mg is less than about 3.3 ng / mg.

[0427] 287. The method of any one of the preceding embodiments after 229, wherein the second ratio less than about 14 ng / mg is less than about 7.3 ng / mg.

[0428] 288. The method of any one of the preceding embodiments after 229, wherein the second ratio less than about 14 ng / mg is less than about 7.2 ng / mg.

[0429] 289. The method of any one of the preceding embodiments after 229, wherein the second ratio less than about 14 ng / mg is less than about 7.1 ng / mg.

[0430] 290. The method of any one of the preceding embodiments after 229, wherein the second ratio less than about 14 ng / mg is less than about 7 ng / mg.

[0431] 291. The method of any one of the preceding embodiments after 229, wherein the second ratio less than about 14 ng / mg is less than about 6 ng / mg.

[0432] 292. The method of any one of the preceding embodiments after 229, wherein the second ratio less than about 14 ng / mg is less than about 5 ng / mg.

[0433] 293. The method of any one of the preceding embodiments after 229, wherein the second ratio less than about 14 ng / mg is less than about 4 ng / mg.

[0434] 294. The method of any one of the preceding embodiments after 229, wherein the second ratio less than about 14 ng / mg is less than about 3.9 ng / mg.

[0435] 295. The method of any one of the preceding embodiments after 229, wherein the second ratio less than about 14 ng / mg is less than about 3.8 ng / mg.

[0436] 296. The method of any one of the preceding embodiments after 229, wherein the second ratio less than about 14 ng / mg is less than about 3.7 ng / mg.

[0437] 297. The method of any one of the preceding embodiments after 229, wherein the second ratio less than about 14 ng / mg is less than about 3.6 ng / mg.

[0438] 298. The method of any one of the preceding embodiments after 229, wherein the second ratio less than about 14 ng / mg is less than about 3.5 ng / mg.

[0439] 299. The method of any one of the preceding embodiments after 229, wherein the higher likelihood of graft failure is about 5% or greater.

[0440] 300. The method of any one of the preceding embodiments after 229, wherein the higher likelihood of graft failure is greater than or equal to about 8%.

[0441] 301. The method of any one of the preceding embodiments after 229, wherein the higher likelihood of graft failure is greater than or equal to about 10%.

[0442] 302. The method of any one of the preceding embodiments after 229, wherein the higher likelihood of graft failure is greater than or equal to about 15%.

[0443] 303. The method of any one of the preceding embodiments after 229, wherein the higher likelihood of graft failure is greater than or equal to about 16%.

[0444] 304. The method of any one of the preceding embodiments after 229, wherein the higher likelihood of graft failure is greater than or equal to about 20%.

[0445] 305. The method of any one of the preceding embodiments after 229, wherein the higher likelihood of graft failure is greater than or equal to about 22%.

[0446] 306. The method of any one of the preceding embodiments after 229, wherein the higher likelihood of graft failure is greater than or equal to about 25%.

[0447] 307. The method of any one of the preceding embodiments after 229, wherein the higher likelihood of graft failure is greater than or equal to about 30%.

[0448] 308. The method of any one of the preceding embodiments after 229, wherein the higher likelihood of graft failure is greater than or equal to about 32%.

[0449] 309. The method of any one of the preceding embodiments after 229, wherein the higher likelihood of graft failure is greater than or equal to about 35%.

[0450] 310. The method of any one of the preceding embodiments after 229, wherein the higher likelihood of graft failure is greater than or equal to about 36%.

[0451] 311. The method of any one of the preceding embodiments after 229, wherein the higher likelihood of graft failure is about 40% or greater.

[0452] 312. The method of any one of the preceding embodiments after 229, wherein the higher likelihood of graft failure is about 45% or greater.

[0453] 313. The method of any one of the preceding embodiments after 229, wherein the higher likelihood of graft failure is greater than or equal to about 50%.

[0454] 314. The method of any one of the preceding embodiments after 229, wherein the higher likelihood of graft dysfunction is between about 5% and about 50%.

[0455] 315. The method of any one of the preceding embodiments after 229, wherein the higher likelihood of graft dysfunction is between about 10% and about 45%.

[0456] 316. The method of any one of the preceding embodiments after 229, wherein the higher likelihood of graft dysfunction is between about 20% and about 40%.

[0457] 317. The method of any one of the preceding embodiments after 229, wherein the higher likelihood of graft failure is about 25% to about 35%.

[0458] 318. The method of any one of the preceding embodiments after 229, wherein the higher likelihood of graft dysfunction is about 25% to about 30%.

[0459] 319. The method of any one of the preceding embodiments after 229, wherein the higher likelihood of graft failure is between about 10% and about 20%.

[0460] 320. The method of any one of the preceding embodiments after 229, wherein the higher likelihood of graft failure is between about 15% and about 25%.

[0461] 321. The method of any one of the preceding embodiments after 229, wherein the higher likelihood of graft dysfunction is about 20% to about 30%.

[0462] 322. The method of any one of the preceding embodiments after 229, wherein the higher likelihood of graft dysfunction is about 30% to about 40%.

[0463] 323. The method of any one of the preceding embodiments after 229, wherein the higher likelihood of graft failure is between about 35% and about 45%.

[0464] 324. The method of any one of the preceding embodiments after 229, wherein the higher likelihood of graft dysfunction is between about 40% and about 50%.

[0465] 325. The method of any one of the preceding embodiments after 229, wherein the graft failure occurs after two years.

[0466] 326. The method of any one of the preceding embodiments after 229, wherein the graft failure occurs after 3 years.

[0467] 327. The method of any one of the preceding embodiments after 229, wherein the graft failure occurs after 4 years.

[0468] 328. The method of any one of the preceding embodiments after 229, wherein the graft failure occurs after 5 years.

[0469] 329. The method of any one of the preceding embodiments after 229, wherein the graft failure occurs after 6 years.

[0470] 330. The method of any one of the preceding embodiments after 229, wherein the graft failure occurs after 7 years.

[0471] 331. The method of any one of the preceding embodiments after 229, wherein the graft failure occurs after 8 years.

[0472] 332. The method of any one of the preceding embodiments after 229, wherein the graft failure occurs after 9 years.

[0473] 333. The method of any one of the preceding embodiments after 229, wherein the graft failure occurs within 7 years.

[0474] 334. The method of any one of the preceding embodiments after 229, wherein the graft failure occurs within 8 years.

[0475] 335. The method of any one of the preceding embodiments after 229, wherein the graft failure occurs within 9 years.

[0476] 336. The method of any one of the preceding embodiments after 229, further comprising assessing the probability of graft failure based on the ratio and at least one marker.

[0477] 337. The method of embodiment 336, wherein the at least one other marker comprises age, sex, time since transplant at enrollment, urinary albumin-to-creatine ratio (UACR), estimated glomerular filtration rate (eGFR) based on the creatinine and cystatin C-based CKD-EPI equation, presence of human leukocyte antigen, or any combination thereof.

[0478] 338. The method of embodiment 337, wherein the assessed probability exhibits an area under the receiver operating characteristic (ROC) curve (AUC) of at least about 70 percent (%).

[0479] 339. The method of embodiment 338, wherein the assessed probability represents at least about 80 percent (%) of the AUC.

[0480] 340. The method of embodiment 338, wherein the assessed probability represents at least about 85 percent (%) of the AUC.

[0481] 341. The method of embodiment 338, wherein the assessed probability represents at least about 90 percent (%) of the AUC.

[0482] 342. The method of embodiment 338, wherein the assessed probability represents at least about 95 percent (%) of the AUC.

[0483] Second Set of Non-Limiting Embodiments The present disclosure is also described by the following second set of non-limiting embodiments. However, the use of these and other embodiments anywhere in the specification is for illustrative purposes only and in no way limits the scope and meaning of the present disclosure. Likewise, the present disclosure is not limited to any particular preferred embodiment or aspect described herein. Indeed, modifications and variations may be apparent to those skilled in the art upon reading this specification, and such variations can be made without departing from the spirit or scope of the present disclosure.

[0484] 1. A method of preparing an assay, comprising: providing a solution comprising a urine sample from a kidney transplant recipient after kidney transplantation and a reagent for interacting with the urinary binding peptide-containing fetuin A fragment; measuring the average excretion of fetuin A fragments into urine from said solution; correlating the measured average fetuin A fragment excretion amount with the likelihood of graft failure in the recipient; a measured average fetuin A fragment excretion rate greater than about 0.8 micrograms per hour (μg / h) indicates a higher likelihood of graft failure over a period of about 10 years or less; said correlating with one another; The method comprising:

[0485] 2. The method of embodiment 1, wherein said excretion rate being greater than about 0.8 μg / h triggers medical intervention to prevent said graft failure.

[0486] 3. The method of any of embodiments 1-2, wherein said graft decline is indicated by a doubling of serum creatinine, the need for kidney retransplantation, or the need for dialysis.

[0487] 4. The recipient has a urine flow rate of approximately 25 milliliters per minute per 1.73 square meters (mL / min / 1.73 m) at a time near the time of collection of the urine sample. 2 4. The method of any one of embodiments 1 to 3, wherein the patient has been shown to have an eGFR of ≥ 100 mg / kg.

[0488] 5. The method of any one of embodiments 1-4, wherein the average urinary excretion of fetuin-A fragments is averaged over a period of about 2 hours or more.

[0489] 6. The method of any one of embodiments 1-4, wherein the average urinary excretion of fetuin-A fragments is averaged over a period of about 4 hours or more.

[0490] 7. The method of any one of embodiments 1-4, wherein the average urinary excretion of fetuin-A fragments is averaged over a period of about 6 hours or more.

[0491] 8. The method of any one of embodiments 1-4, wherein the average urinary excretion of fetuin-A fragments is averaged over a period of about 12 hours or more.

[0492] 9. The method of any one of embodiments 1-4, wherein the average urinary excretion of fetuin-A fragments is averaged over a period of about 24 hours or more.

[0493] 10. The method of any one of embodiments 1-4, wherein the average urinary excretion of fetuin-A fragments is averaged over a period of about 36 hours or more.

[0494] 11. The method of any one of embodiments 1-4, wherein the average urinary excretion of fetuin-A fragments is averaged over a period of about 48 hours or more.

[0495] 12. The method of any one of embodiments 1-11, wherein the urine sample is collected from the recipient of the kidney transplant at least about 1 day after the kidney transplant.

[0496] 13. The method of any one of embodiments 1-11, wherein the urine sample is collected from the recipient of the kidney transplant at least about 2 days after the kidney transplant.

[0497] 14. The method of any one of embodiments 1-11, wherein the urine sample is collected from the recipient of the kidney transplant at least about 1 week after the kidney transplant.

[0498] 15. The method of any one of embodiments 1 to 11, wherein the urine sample is collected from the recipient of the kidney transplant at least about 2 weeks after the kidney transplant.

[0499] 16. The method of any one of embodiments 1 to 11, wherein the urine sample is collected from the recipient of the kidney transplant at least about one month after the kidney transplant.

[0500] 17. The method of any one of embodiments 1 to 11, wherein the urine sample is collected from the recipient of the kidney transplant at least about 3 months after the kidney transplant.

[0501] 18. The method of any one of embodiments 1 to 11, wherein the urine sample is collected from the recipient of the kidney transplant at least about 6 months after the kidney transplant.

[0502] 19. The method of any one of embodiments 1 to 11, wherein the urine sample is collected from the recipient of the kidney transplant at least about 12 months after the kidney transplant.

[0503] 20. The method of any one of embodiments 1 to 11, wherein the urine sample is collected from the recipient of the kidney transplant at least about 24 months after the kidney transplant.

[0504] 21. The method of any one of embodiments 1-20, wherein the average fetuin A fragment excretion rate greater than about 0.8 μg / h indicates a higher likelihood of graft function decline of the transplanted kidney over a period of about 9 years or less.

[0505] 22. The method of any one of embodiments 1-20, wherein the average fetuin A fragment excretion rate greater than about 0.8 μg / h indicates a higher likelihood of graft function decline of the transplanted kidney over a period of about 7 years or less.

[0506] 23. The method of any one of embodiments 1 to 20, wherein the average fetuin A fragment excretion rate greater than about 0.8 μg / h indicates a higher likelihood of graft function decline in the transplanted kidney over a period of about 5 years or less.

[0507] 24. The method of any one of embodiments 1 to 20, wherein the average fetuin A fragment excretion rate greater than about 0.8 μg / h indicates a higher likelihood of graft function decline in the transplanted kidney over a period of about 3 years or less.

[0508] 25. The method of any one of embodiments 1 to 20, wherein the average fetuin A fragment excretion rate greater than about 0.8 μg / h indicates a higher likelihood of graft function decline in the transplanted kidney over a period of about 1 year or less.

[0509] 26. The method of any one of embodiments 1 to 25, wherein the average fetuin A fragment excretion rate greater than about 0.8 μg / h is greater than about 0.9 μg / h.

[0510] 27. The method of any one of embodiments 1 to 25, wherein the average fetuin A fragment excretion rate greater than about 0.8 μg / h is greater than about 1 μg / h.

[0511] 28. The method of any one of embodiments 1-25, wherein the average fetuin A fragment excretion rate greater than about 0.8 μg / h is greater than about 2 μg / h.

[0512] 29. The method of any one of embodiments 1 to 25, wherein the average fetuin A fragment excretion rate greater than about 0.8 μg / h is greater than about 2.1 μg / h.

[0513] 30. The method of any one of embodiments 1-25, wherein the average fetuin A fragment excretion rate greater than about 0.8 μg / h is greater than about 2.2 μg / h.

[0514] 31. The method of any one of embodiments 1-25, wherein the average fetuin A fragment excretion rate greater than about 0.8 μg / h is greater than about 2.3 μg / h.

[0515] 32. The method of any one of embodiments 1-25, wherein the average fetuin A fragment excretion rate greater than about 0.8 μg / h is greater than about 2.5 μg / h.

[0516] 33. A method according to any one of embodiments 1 to 32, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 0.9 μg / h to about 2.4 μg / h and the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 0.9 μg / h or less is about 2 or greater.

[0517] 34. A method described in any one of embodiments 1 to 32, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate being between about 0.9 μg / h and about 2.4 μg / h and the average fetuin A fragment excretion rate being less than about 0.9 μg / h is about 1.5 or greater.

[0518] 35. A method described in any one of embodiments 1 to 32, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate being between about 0.9 μg / h and about 2.4 μg / h and the average fetuin A fragment excretion rate being less than about 0.9 μg / h is about 1.59 or greater.

[0519] 36. A method described in any one of embodiments 1 to 32, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate being between about 0.9 μg / h and about 2.4 μg / h and the average fetuin A fragment excretion rate being less than about 0.9 μg / h is about 1.6 or greater.

[0520] 37. A method described in any one of embodiments 1 to 32, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate being between about 0.9 μg / h and about 2.4 μg / h and the average fetuin A fragment excretion rate being less than about 0.9 μg / h is about 1.7 or greater.

[0521] 38. A method described in any one of embodiments 1 to 32, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate being between about 0.9 μg / h and about 2.4 μg / h and the average fetuin A fragment excretion rate being less than about 0.9 μg / h is about 1.78 or greater.

[0522] 39. A method described in any one of embodiments 1 to 32, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate being between about 0.9 μg / h and about 2.4 μg / h and the average fetuin A fragment excretion rate being less than about 0.9 μg / h is about 1.79 or greater.

[0523] 40. A method described in any one of embodiments 1 to 32, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate being between about 0.9 μg / h and about 2.4 μg / h and the average fetuin A fragment excretion rate being less than about 0.9 μg / h is about 1.8 or greater.

[0524] 41. A method described in any one of embodiments 1 to 32, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate being between about 0.9 μg / h and about 2.4 μg / h and the average fetuin A fragment excretion rate being less than about 0.9 μg / h is about 2.1 or greater.

[0525] 42. A method according to any one of embodiments 1 to 32, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate being between about 0.9 μg / h and about 2.4 μg / h and the average fetuin A fragment excretion rate being less than about 0.9 μg / h is about 2.15 or greater.

[0526] 43. A method according to any one of embodiments 1 to 32, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate being between about 0.9 μg / h and about 2.4 μg / h and the average fetuin A fragment excretion rate being less than or equal to about 0.9 μg / h is about 2.18 or greater.

[0527] 44. A method described in any one of embodiments 1 to 32, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate being between about 0.9 μg / h and about 2.4 μg / h and the average fetuin A fragment excretion rate being less than about 0.9 μg / h is about 2.2 or greater.

[0528] 45. A method according to any one of embodiments 1 to 32, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate being between about 0.9 μg / h and about 2.4 μg / h and the average fetuin A fragment excretion rate being less than about 0.9 μg / h is about 2.22 or greater.

[0529] 46. ​​A method described in any one of embodiments 1 to 32, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate being between about 0.9 μg / h and about 2.4 μg / h and the average fetuin A fragment excretion rate being less than about 0.9 μg / h is between about 1.5 and about 2.5.

[0530] 47. A method according to any one of embodiments 1 to 32, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate being between about 0.9 μg / h and about 2.4 μg / h and the average fetuin A fragment excretion rate being less than about 0.9 μg / h is between about 1.6 and about 2.3.

[0531] 48. A method according to any one of embodiments 1 to 32, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate being between about 0.9 μg / h and about 2.4 μg / h and the average fetuin A fragment excretion rate being less than about 0.9 μg / h is between about 1.59 and about 2.22.

[0532] 49. A method according to any one of embodiments 1 to 48, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 2.4 μg / h or more and the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 0.9 μg / h or less is about 2 or more.

[0533] 50. A method described in any one of embodiments 1 to 48, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 2.4 μg / h or more and the average fetuin A fragment excretion rate of about 0.9 μg / h or less is about 2.2 or more.

[0534] 51. A method described in any one of embodiments 1 to 48, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 2.4 μg / h or more and the average fetuin A fragment excretion rate of about 0.9 μg / h or less is about 2.26 or more.

[0535] 52. A method described in any one of embodiments 1 to 48, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 2.4 μg / h or more and the average fetuin A fragment excretion rate of about 0.9 μg / h or less is about 2.5 or more.

[0536] 53. A method described in any one of embodiments 1 to 48, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 2.4 μg / h or more and the average fetuin A fragment excretion rate of about 0.9 μg / h or less is about 2.7 or more.

[0537] 54. A method described in any one of embodiments 1 to 48, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 2.4 μg / h or more and the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 0.9 μg / h or less is about 3 or more.

[0538] 55. A method described in any one of embodiments 1 to 48, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 2.4 μg / h or more and the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 0.9 μg / h or less is about 3.5 or more.

[0539] 56. A method described in any one of embodiments 1 to 48, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 2.4 μg / h or more and the average fetuin A fragment excretion rate of about 0.9 μg / h or less is about 3.6 or more.

[0540] 57. A method described in any one of embodiments 1 to 48, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 2.4 μg / h or more and the average fetuin A fragment excretion rate of about 0.9 μg / h or less is about 3.7 or more.

[0541] 58. A method according to any one of embodiments 1 to 48, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 2.4 μg / h or more and the average fetuin A fragment excretion rate of about 0.9 μg / h or less is about 3.7 or more.

[0542] 59. A method described in any one of embodiments 1 to 48, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 2.4 μg / h or more and the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 0.9 μg / h or less is about 4 or more.

[0543] 60. A method described in any one of embodiments 1 to 48, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 2.4 μg / h or more and the average fetuin A fragment excretion rate of about 0.9 μg / h or less is about 4.5 or more.

[0544] 61. A method described in any one of embodiments 1 to 48, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 2.4 μg / h or more and the average fetuin A fragment excretion rate of about 0.9 μg / h or less is about 4.7 or more.

[0545] 62. A method described in any one of embodiments 1 to 48, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 2.4 μg / h or more and the average fetuin A fragment excretion rate of about 0.9 μg / h or less is about 4.8 or more.

[0546] 63. A method according to any one of embodiments 1 to 48, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 2.4 μg / h or more and the average fetuin A fragment excretion rate of about 0.9 μg / h or less is about 4.9 or more.

[0547] 64. A method described in any one of embodiments 1 to 48, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 2.4 μg / h or more and the average fetuin A fragment excretion rate of about 0.9 μg / h or less is about 5 or more.

[0548] 65. A method according to any one of embodiments 1 to 48, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 2.4 μg / h or more and the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 0.9 μg / h or less is about 2 to about 5.

[0549] 66. A method according to any one of embodiments 1 to 48, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 2.4 μg / h or more and the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 0.9 μg / h or less is about 2 to about 5.

[0550] 67. A method according to any one of embodiments 1 to 48, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 2.4 μg / h or more and the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 0.9 μg / h or less is about 2.5 to about 5.

[0551] 68. A method according to any one of embodiments 1 to 48, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 2.4 μg / h or more and the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 0.9 μg / h or less is about 3.5 to about 5.

[0552] 69. The method of any preceding embodiment, wherein said interacting with said fetuin A fragment comprises binding to said fetuin A.

[0553] 70. The method of any one of embodiments 1-68, wherein the reagent comprises an antibody.

[0554] 71. An assay kit for determining the possibility of graft dysfunction in a transplanted kidney, comprising: a solution comprising a reagent at a concentration sufficient to interact with the amount of urinary binding peptide-containing fetuin A fragment in a urine sample from a kidney transplant recipient; a device for measuring an average excretion amount of fetuin-A fragments into urine from said solution and correlating said measured average excretion amount of fetuin-A fragments with a likelihood of graft failure in said recipient, a measured average fetuin A fragment excretion rate greater than about 0.8 micrograms per hour (μg / h) indicates a higher likelihood of graft failure over a period of about 10 years or less; the device; The assay kit comprises:

[0555] 72. The assay kit of embodiment 71, wherein said excretion rate being greater than about 0.8 μg / h triggers medical intervention to prevent said graft failure.

[0556] 73. The assay kit of any of embodiments 71-72, wherein said graft decline is indicated by a doubling of serum creatinine, the need for kidney retransplantation, or the need for dialysis.

[0557] 74. The recipient has a urinary tract infection rate of approximately 25 milliliters per minute per 1.73 square meters (mL / min / 1.73 m) at a time near the time of collection of the urine specimen. 2 74. The assay kit of any of embodiments 71 to 73, wherein the patient has been shown to have an eGFR of 100 or more.

[0558] 75. The assay kit of any of embodiments 71-74, wherein the average urinary excretion of fetuin-A fragments is averaged over a period of about 2 hours or more.

[0559] 76. The assay kit of any of embodiments 71-74, wherein the average urinary excretion of fetuin-A fragments is averaged over a period of about 4 hours or more.

[0560] 77. The assay kit of any of embodiments 71-74, wherein the average urinary excretion of fetuin A fragments is averaged over a period of about 6 hours or more.

[0561] 78. The assay kit of any of embodiments 71-74, wherein the average urinary excretion of fetuin A fragments is averaged over a period of about 12 hours or more.

[0562] 79. The assay kit of any of embodiments 71-74, wherein the average urinary excretion of fetuin A fragments is averaged over a period of about 24 hours or more.

[0563] 80. The assay kit of any of embodiments 71-74, wherein the average urinary excretion of fetuin A fragments is averaged over a period of about 36 hours or more.

[0564] 81. The assay kit of any of embodiments 71-74, wherein the average urinary excretion of fetuin A fragments is averaged over a period of about 48 hours or more.

[0565] 82. The assay kit of any of embodiments 71-81, wherein the urine sample is collected from the recipient of the kidney transplant at least about 1 day after the kidney transplant.

[0566] 83. The assay kit of any of embodiments 71-81, wherein the urine sample is collected from the recipient of the kidney transplant at least about 2 days after the kidney transplant.

[0567] 84. The assay kit of any of embodiments 71-81, wherein the urine sample is collected from the recipient of the kidney transplant at least about one week after the kidney transplant.

[0568] 85. An assay kit according to any of embodiments 71 to 81, wherein the urine sample is collected from the recipient of the kidney transplant at least about two weeks after the kidney transplant.

[0569] 86. An assay kit according to any of embodiments 71 to 81, wherein the urine sample is collected from the recipient of the kidney transplant at least about one month after the kidney transplant.

[0570] 87. An assay kit according to any of embodiments 71 to 81, wherein the urine sample is collected from the recipient of the kidney transplant at least about 3 months after the kidney transplant.

[0571] 88. An assay kit according to any of embodiments 71 to 81, wherein the urine sample is collected from the recipient of the kidney transplant at least about 6 months after the kidney transplant.

[0572] 89. An assay kit according to any of embodiments 71 to 81, wherein the urine sample is collected from the recipient of the kidney transplant at least about 12 months after the kidney transplant.

[0573] 90. The assay kit of any of embodiments 71-81, wherein the urine sample is collected from the recipient of the kidney transplant at least about 24 months after the kidney transplant.

[0574] 91. An assay kit described in any of embodiments 71 to 90, wherein an average fetuin A fragment excretion rate higher than about 0.8 μg / h indicates a higher likelihood of graft function decline in the transplanted kidney over a period of about 9 years or less.

[0575] 92. An assay kit according to any one of embodiments 71 to 90, wherein the average fetuin A fragment excretion rate greater than about 0.8 μg / h indicates a higher likelihood of graft function decline in the transplanted kidney over a period of about 7 years or less.

[0576] 93. An assay kit according to any one of embodiments 71 to 90, wherein the average fetuin A fragment excretion rate greater than about 0.8 μg / h indicates a higher likelihood of graft function decline in the transplanted kidney over a period of about 5 years or less.

[0577] 94. An assay kit according to any one of embodiments 71 to 90, wherein the average fetuin A fragment excretion rate greater than about 0.8 μg / h indicates a higher likelihood of graft function decline in the transplanted kidney over a period of about 3 years or less.

[0578] 95. An assay kit according to any one of embodiments 71 to 90, wherein the average fetuin A fragment excretion rate greater than about 0.8 μg / h indicates a higher likelihood of graft function decline in the transplanted kidney over a period of about one year or less.

[0579] 96. The assay kit of any of embodiments 71-95, wherein the average fetuin A fragment excretion rate greater than about 0.8 μg / h is greater than about 0.9 μg / h.

[0580] 97. The assay kit of any of embodiments 71-95, wherein the average fetuin A fragment excretion rate greater than about 0.8 μg / h is greater than about 1 μg / h.

[0581] 98. The assay kit of any of embodiments 71-95, wherein the average fetuin A fragment excretion rate greater than about 0.8 μg / h is greater than about 2 μg / h.

[0582] 99. The assay kit of any of embodiments 71-95, wherein the average fetuin A fragment excretion rate greater than about 0.8 μg / h is greater than about 2.1 μg / h.

[0583] 100. The assay kit of any of embodiments 71-95, wherein the average fetuin A fragment excretion rate greater than about 0.8 μg / h is greater than about 2.2 μg / h.

[0584] 101. The assay kit of any of embodiments 71-95, wherein the average fetuin A fragment excretion rate greater than about 0.8 μg / h is greater than about 2.3 μg / h.

[0585] 102. The assay kit of any of embodiments 71-95, wherein the average fetuin A fragment excretion rate greater than about 0.8 μg / h is greater than about 2.5 μg / h.

[0586] 103. An assay kit described in any of embodiments 71 to 102, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 0.9 μg / h to about 2.4 μg / h and the average fetuin A fragment excretion rate of about 0.9 μg / h or less is about 2 or greater.

[0587] 104. An assay kit described in any of embodiments 71 to 102, wherein the hazard ratio corresponding to the average fetuin A fragment excretion amount being between about 0.9 μg / h and about 2.4 μg / h and the average fetuin A fragment excretion amount being less than about 0.9 μg / h is about 1.5 or more.

[0588] 105. An assay kit described in any of embodiments 71 to 102, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 0.9 μg / h to about 2.4 μg / h and the average fetuin A fragment excretion rate of about 0.9 μg / h or less is about 1.59 or greater.

[0589] 106. An assay kit described in any of embodiments 71 to 102, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 0.9 μg / h to about 2.4 μg / h and the average fetuin A fragment excretion rate of about 0.9 μg / h or less is about 1.6 or greater.

[0590] 107. An assay kit described in any of embodiments 71 to 102, wherein the hazard ratio corresponding to the average fetuin A fragment excretion amount being between about 0.9 μg / h and about 2.4 μg / h and the average fetuin A fragment excretion amount being less than about 0.9 μg / h is about 1.7 or greater.

[0591] 108. An assay kit described in any of embodiments 71 to 102, wherein the hazard ratio corresponding to the average fetuin A fragment excretion amount being between about 0.9 μg / h and about 2.4 μg / h and the average fetuin A fragment excretion amount being less than about 0.9 μg / h is about 1.78 or greater.

[0592] 109. An assay kit described in any of embodiments 71 to 102, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate being between about 0.9 μg / h and about 2.4 μg / h and the average fetuin A fragment excretion rate being less than about 0.9 μg / h is about 1.79 or greater.

[0593] 110. An assay kit described in any of embodiments 71 to 102, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate being between about 0.9 μg / h and about 2.4 μg / h and the average fetuin A fragment excretion rate being less than about 0.9 μg / h is about 1.8 or greater.

[0594] 111. An assay kit described in any of embodiments 71 to 102, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate being between about 0.9 μg / h and about 2.4 μg / h and the average fetuin A fragment excretion rate being less than about 0.9 μg / h is about 2.1 or greater.

[0595] 112. An assay kit described in any of embodiments 71 to 102, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate being between about 0.9 μg / h and about 2.4 μg / h and the average fetuin A fragment excretion rate being less than about 0.9 μg / h is about 2.15 or greater.

[0596] 113. An assay kit described in any of embodiments 71 to 102, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate being between about 0.9 μg / h and about 2.4 μg / h and the average fetuin A fragment excretion rate being less than about 0.9 μg / h is about 2.18 or greater.

[0597] 114. An assay kit described in any of embodiments 71 to 102, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate being between about 0.9 μg / h and about 2.4 μg / h and the average fetuin A fragment excretion rate being less than about 0.9 μg / h is about 2.2 or greater.

[0598] 115. An assay kit described in any of embodiments 71 to 102, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 0.9 μg / h to about 2.4 μg / h and the average fetuin A fragment excretion rate of about 0.9 μg / h or less is about 2.22 or greater.

[0599] 116. An assay kit described in any of embodiments 71 to 102, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate being about 0.9 μg / h to about 2.4 μg / h and the average fetuin A fragment excretion rate being less than about 0.9 μg / h is about 1.5 to about 2.5.

[0600] 117. An assay kit described in any of embodiments 71 to 102, wherein the hazard ratio corresponding to the average fetuin A fragment excretion amount being about 0.9 μg / h to about 2.4 μg / h and the average fetuin A fragment excretion amount being less than about 0.9 μg / h is about 1.6 to about 2.3.

[0601] 118. An assay kit described in any of embodiments 71 to 102, wherein the hazard ratio corresponding to the average fetuin A fragment excretion amount being about 0.9 μg / h to about 2.4 μg / h and the average fetuin A fragment excretion amount being less than about 0.9 μg / h is about 1.59 to about 2.22.

[0602] 119. An assay kit described in any of embodiments 71 to 117, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 2.4 μg / h or more and the average fetuin A fragment excretion rate of about 0.9 μg / h or less is about 2 or more.

[0603] 120. An assay kit described in any of embodiments 71 to 117, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 2.4 μg / h or more and the average fetuin A fragment excretion rate of about 0.9 μg / h or less is about 2.2 or more.

[0604] 121. An assay kit described in any of embodiments 71 to 117, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 2.4 μg / h or more and the average fetuin A fragment excretion rate of about 0.9 μg / h or less is about 2.26 or more.

[0605] 122. An assay kit described in any of embodiments 71 to 117, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 2.4 μg / h or more and the average fetuin A fragment excretion rate of about 0.9 μg / h or less is about 2.5 or more.

[0606] 123. An assay kit described in any of embodiments 71 to 117, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 2.4 μg / h or more and the average fetuin A fragment excretion rate of about 0.9 μg / h or less is about 2.7 or more.

[0607] 124. An assay kit described in any of embodiments 71 to 117, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 2.4 μg / h or more and the average fetuin A fragment excretion rate of about 0.9 μg / h or less is about 3 or more.

[0608] 125. An assay kit described in any of embodiments 71 to 117, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 2.4 μg / h or more and the average fetuin A fragment excretion rate of about 0.9 μg / h or less is about 3.5 or more.

[0609] 126. An assay kit described in any of embodiments 71 to 117, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 2.4 μg / h or more and the average fetuin A fragment excretion rate of about 0.9 μg / h or less is about 3.6 or more.

[0610] 127. An assay kit described in any of embodiments 71 to 117, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 2.4 μg / h or more and the average fetuin A fragment excretion rate of about 0.9 μg / h or less is about 3.7 or more.

[0611] 128. An assay kit described in any of embodiments 71 to 117, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 2.4 μg / h or more and the average fetuin A fragment excretion rate of about 0.9 μg / h or less is about 3.7 or more.

[0612] 129. An assay kit described in any of embodiments 71 to 117, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 2.4 μg / h or more and the average fetuin A fragment excretion rate of about 0.9 μg / h or less is about 4 or more.

[0613] 130. An assay kit described in any of embodiments 71 to 117, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 2.4 μg / h or more and the average fetuin A fragment excretion rate of about 0.9 μg / h or less is about 4.5 or more.

[0614] 131. An assay kit described in any of embodiments 71 to 117, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 2.4 μg / h or more and the average fetuin A fragment excretion rate of about 0.9 μg / h or less is about 4.7 or more.

[0615] 132. An assay kit described in any of embodiments 71 to 117, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 2.4 μg / h or more and the average fetuin A fragment excretion rate of about 0.9 μg / h or less is about 4.8 or more.

[0616] 133. An assay kit described in any of embodiments 71 to 117, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 2.4 μg / h or more and the average fetuin A fragment excretion rate of about 0.9 μg / h or less is about 4.9 or more.

[0617] 134. An assay kit described in any of embodiments 71 to 117, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 2.4 μg / h or more and the average fetuin A fragment excretion rate of about 0.9 μg / h or less is about 5 or more.

[0618] 135. An assay kit described in any of embodiments 71 to 117, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 2.4 μg / h or more and the average fetuin A fragment excretion rate of about 0.9 μg / h or less is about 2 to about 5.

[0619] 136. An assay kit described in any of embodiments 71 to 117, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 2.4 μg / h or more and the average fetuin A fragment excretion rate of about 0.9 μg / h or less is about 2 to about 5.

[0620] 137. An assay kit described in any of embodiments 71 to 117, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 2.4 μg / h or more and the average fetuin A fragment excretion rate of about 0.9 μg / h or less is about 2.5 to about 5.

[0621] 138. An assay kit described in any of embodiments 71 to 117, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 2.4 μg / h or more and the average fetuin A fragment excretion rate of about 0.9 μg / h or less is about 3.5 to about 5.

[0622] 139. The assay kit of any of embodiments 71-138, wherein said interacting with said fetuin A fragment comprises binding to said fetuin A.

[0623] 140. The assay kit of any of embodiments 71-138, wherein the reagent comprises an antibody.

[0624] 141. A method for correlating the likelihood of graft function decline in a kidney transplant recipient, comprising: obtaining a solution comprising a urine sample from a kidney transplant recipient after kidney transplantation and a reagent for interacting with the urinary binding peptide-containing fetuin A fragment; measuring the average excretion of fetuin A fragments into urine from said solution; correlating the measured average fetuin A fragment excretion amount with the likelihood of graft failure in the recipient; a measured average fetuin A fragment excretion rate greater than about 0.8 micrograms per hour (μg / h) indicates a higher likelihood of graft failure over a period of about 10 years or less; said correlating with one another; The method comprising:

[0625] 142. The method of embodiment 141, wherein the excretion rate is greater than about 0.8 μg / h, triggering medical intervention to prevent graft failure.

[0626] 143. The method of any of embodiments 141-142, wherein the graft decline is indicated by a doubling of serum creatinine, the need for kidney retransplantation, or the need for dialysis.

[0627] 144. The recipient has a urine flow rate of approximately 25 milliliters per minute per 1.73 square meters (mL / min / 1.73 m) at a time near the time of collection of the urine sample. 2 144. The method of any of embodiments 141-143, wherein the patient has been shown to have an eGFR of ≥ 100 mg / kg.

[0628] 145. The method of any of embodiments 141-144, wherein the average urinary excretion of fetuin-A fragments is averaged over a period of about 2 hours or more.

[0629] 146. The method of any of embodiments 141-144, wherein the average urinary excretion of fetuin-A fragments is averaged over a period of about 4 hours or more.

[0630] 147. The method of any of embodiments 141-144, wherein the average urinary excretion of fetuin-A fragments is averaged over a period of about 6 hours or more.

[0631] 148. The method of any of embodiments 141-144, wherein the average urinary excretion of fetuin-A fragments is averaged over a period of about 12 hours or more.

[0632] 149. The method of any of embodiments 141-144, wherein the average urinary excretion of fetuin-A fragments is averaged over a period of about 24 hours or more.

[0633] 150. The method of any of embodiments 141-144, wherein the average urinary excretion of fetuin-A fragments is averaged over a period of about 36 hours or more.

[0634] 151. The method of any of embodiments 141-144, wherein the average urinary excretion of fetuin-A fragments is averaged over a period of about 48 hours or more.

[0635] 152. The method of any of embodiments 141-151, wherein the urine sample is collected from the recipient of the kidney transplant at least about 1 day after the kidney transplant.

[0636] 153. The method of any of embodiments 141-151, wherein the urine sample is collected from the recipient of the kidney transplant at least about 2 days after the kidney transplant.

[0637] 154. The method of any of embodiments 141-151, wherein the urine sample is collected from the recipient of the kidney transplant at least about 1 week after the kidney transplant.

[0638] 155. The method of any of embodiments 141-151, wherein the urine sample is collected from the recipient of the kidney transplant at least about 2 weeks after the kidney transplant.

[0639] 156. The method of any of embodiments 141 to 151, wherein the urine sample is collected from the recipient of the kidney transplant at least about one month after the kidney transplant.

[0640] 157. The method of any of embodiments 141-151, wherein the urine sample is collected from the recipient of the kidney transplant at least about 3 months after the kidney transplant.

[0641] 158. The method of any of embodiments 141 to 151, wherein the urine sample is collected from the recipient of the kidney transplant at least about 6 months after the kidney transplant.

[0642] 159. The method of any of embodiments 141 to 151, wherein the urine sample is collected from the recipient of the kidney transplant at least about 12 months after the kidney transplant.

[0643] 160. The method of any of embodiments 141-151, wherein the urine sample is collected from the recipient of the kidney transplant at least about 24 months after the kidney transplant.

[0644] 161. The method of any of embodiments 141-160, wherein the average fetuin A fragment excretion rate higher than about 0.8 μg / h indicates a higher likelihood of graft function decline of the transplanted kidney over a period of about 9 years or less.

[0645] 162. The method of any of embodiments 141-160, wherein the average fetuin A fragment excretion rate higher than about 0.8 μg / h indicates a higher likelihood of graft function decline of the transplanted kidney over a period of about 7 years or less.

[0646] 163. The method of any of embodiments 141-160, wherein the average fetuin A fragment excretion rate greater than about 0.8 μg / h indicates a higher likelihood of graft function decline of the transplanted kidney over a period of about 5 years or less.

[0647] 164. The method of any of embodiments 141-160, wherein the average fetuin A fragment excretion rate higher than about 0.8 μg / h indicates a higher likelihood of graft function decline of the transplanted kidney over a period of about 3 years or less.

[0648] 165. The method of any of embodiments 141-160, wherein the average fetuin A fragment excretion rate higher than about 0.8 μg / h indicates a higher likelihood of graft function decline of the transplanted kidney over a period of about 1 year or less.

[0649] 166. The method of any of embodiments 141-165, wherein the average fetuin A fragment excretion rate greater than about 0.8 μg / h is greater than about 0.9 μg / h.

[0650] 167. The method of any of embodiments 141-165, wherein the average fetuin A fragment excretion rate greater than about 0.8 μg / h is greater than about 1 μg / h.

[0651] 168. The method of any of embodiments 141-165, wherein the average fetuin A fragment excretion rate greater than about 0.8 μg / h is greater than about 2 μg / h.

[0652] 169. The method of any of embodiments 141-165, wherein the average fetuin A fragment excretion rate greater than about 0.8 μg / h is greater than about 2.1 μg / h.

[0653] 170. The method of any of embodiments 141-165, wherein the average fetuin A fragment excretion rate greater than about 0.8 μg / h is greater than about 2.2 μg / h.

[0654] 171. The method of any of embodiments 141-165, wherein the average fetuin A fragment excretion rate greater than about 0.8 μg / h is greater than about 2.3 μg / h.

[0655] 172. The method of any of embodiments 141-165, wherein the average fetuin A fragment excretion rate greater than about 0.8 μg / h is greater than about 2.5 μg / h.

[0656] 173. The method of any of embodiments 141-172, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 0.9 μg / h to about 2.4 μg / h and the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 0.9 μg / h or less is about 2 or greater.

[0657] 174. A method according to any one of embodiments 141 to 172, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 0.9 μg / h to about 2.4 μg / h and the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 0.9 μg / h or less is about 1.5 or greater.

[0658] 175. A method according to any one of embodiments 141 to 172, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 0.9 μg / h to about 2.4 μg / h and the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 0.9 μg / h or less is about 1.59 or greater.

[0659] 176. A method according to any one of embodiments 141 to 172, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 0.9 μg / h to about 2.4 μg / h and the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 0.9 μg / h or less is about 1.6 or greater.

[0660] 177. A method according to any one of embodiments 141 to 172, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 0.9 μg / h to about 2.4 μg / h and the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 0.9 μg / h or less is about 1.7 or greater.

[0661] 178. A method according to any one of embodiments 141 to 172, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 0.9 μg / h to about 2.4 μg / h and the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 0.9 μg / h or less is about 1.78 or greater.

[0662] 179. A method according to any one of embodiments 141 to 172, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 0.9 μg / h to about 2.4 μg / h and the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 0.9 μg / h or less is about 1.79 or greater.

[0663] 180. The method of any of embodiments 141 to 172, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 0.9 μg / h to about 2.4 μg / h and the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 0.9 μg / h or less is about 1.8 or greater.

[0664] 181. The method of any of embodiments 141 to 172, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 0.9 μg / h to about 2.4 μg / h and the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 0.9 μg / h or less is about 2.1 or greater.

[0665] 182. The method of any of embodiments 141 to 172, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 0.9 μg / h to about 2.4 μg / h and the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 0.9 μg / h or less is about 2.15 or greater.

[0666] 183. The method of any of embodiments 141-172, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 0.9 μg / h to about 2.4 μg / h and the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 0.9 μg / h or less is about 2.18 or greater.

[0667] 184. The method of any of embodiments 141 to 172, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 0.9 μg / h to about 2.4 μg / h and the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 0.9 μg / h or less is about 2.2 or greater.

[0668] 185. The method of any of embodiments 141 to 172, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 0.9 μg / h to about 2.4 μg / h and the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 0.9 μg / h or less is about 2.22 or greater.

[0669] 186. The method of any of embodiments 141 to 172, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate being between about 0.9 μg / h and about 2.4 μg / h and the average fetuin A fragment excretion rate being less than or equal to about 0.9 μg / h is between about 1.5 and about 2.5.

[0670] 187. The method of any of embodiments 141 to 172, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate being between about 0.9 μg / h and about 2.4 μg / h and the average fetuin A fragment excretion rate being less than about 0.9 μg / h is between about 1.6 and about 2.3.

[0671] 188. The method of any of embodiments 141 to 172, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate being between about 0.9 μg / h and about 2.4 μg / h and the hazard ratio corresponding to the average fetuin A fragment excretion rate of less than or equal to about 0.9 μg / h is between about 1.59 and about 2.22.

[0672] 189. A method according to any one of embodiments 141 to 188, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 2.4 μg / h or more and the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 0.9 μg / h or less is about 2 or more.

[0673] 190. The method of any of embodiments 141 to 188, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 2.4 μg / h or more and the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 0.9 μg / h or less is about 2.2 or more.

[0674] 191. The method of any of embodiments 141 to 188, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 2.4 μg / h or more and the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 0.9 μg / h or less is about 2.26 or more.

[0675] 192. The method of any of embodiments 141-188, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 2.4 μg / h or more and the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 0.9 μg / h or less is about 2.5 or more.

[0676] 193. The method of any of embodiments 141-188, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 2.4 μg / h or more and the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 0.9 μg / h or less is about 2.7 or more.

[0677] 194. The method of any of embodiments 141 to 188, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 2.4 μg / h or more and the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 0.9 μg / h or less is about 3 or more.

[0678] 195. The method of any of embodiments 141 to 188, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 2.4 μg / h or more and the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 0.9 μg / h or less is about 3.5 or more.

[0679] 196. The method of any of embodiments 141-188, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 2.4 μg / h or more and the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 0.9 μg / h or less is about 3.6 or more.

[0680] 197. The method of any of embodiments 141 to 188, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 2.4 μg / h or more and the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 0.9 μg / h or less is about 3.7 or more.

[0681] 198. The method of any of embodiments 141 to 188, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 2.4 μg / h or more and the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 0.9 μg / h or less is about 3.7 or more.

[0682] 199. A method according to any one of embodiments 141 to 188, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 2.4 μg / h or more and the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 0.9 μg / h or less is about 4 or more.

[0683] 200. A method according to any one of embodiments 141 to 188, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 2.4 μg / h or more and the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 0.9 μg / h or less is about 4.5 or more.

[0684] 201. A method according to any one of embodiments 141 to 188, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 2.4 μg / h or more and the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 0.9 μg / h or less is about 4.7 or more.

[0685] 202. A method according to any one of embodiments 141 to 188, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 2.4 μg / h or more and the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 0.9 μg / h or less is about 4.8 or more.

[0686] 203. A method according to any one of embodiments 141 to 188, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 2.4 μg / h or more and the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 0.9 μg / h or less is about 4.9 or more.

[0687] 204. A method according to any one of embodiments 141 to 188, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 2.4 μg / h or more and the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 0.9 μg / h or less is about 5 or more.

[0688] 205. The method of any of embodiments 141 to 188, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 2.4 μg / h or more and the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 0.9 μg / h or less is about 2 to about 5.

[0689] 206. The method of any of embodiments 141 to 188, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 2.4 μg / h or more and the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 0.9 μg / h or less is about 2 to about 5.

[0690] 207. A method according to any one of embodiments 141 to 188, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 2.4 μg / h or more and the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 0.9 μg / h or less is about 2.5 to about 5.

[0691] 208. A method according to any one of embodiments 141 to 188, wherein the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 2.4 μg / h or more and the hazard ratio corresponding to the average fetuin A fragment excretion rate of about 0.9 μg / h or less is about 3.5 to about 5.

[0692] 209. The method of any of embodiments 141-208, wherein said interacting with said fetuin A fragment comprises binding to said fetuin A.

[0693] 210. The method of any of embodiments 141-208, wherein the reagent comprises an antibody.

[0694] Non-limiting Examples The following examples are provided to illustrate selected embodiments. The examples should not be considered as limiting the scope of the invention, but merely as illustrative and representative thereof. Thus, the examples provided below, although illustrated with specific medical devices or active agents, are applicable to the range of medical devices and active agents described herein.

[0695] Example 1-1 The objective of the clinical performance studies presented in Examples 1-5 is to evaluate the prognostic performance of the Human uPTM3-DKD ELISA test for graft failure in kidney transplant recipients (KTRs). Design and study population

[0696] Design and study population Data were used from the TransplantLines Food and Nutrition Biobank and Cohort Study (NCT03272841). All adult KTRs who visited the outpatient clinic at the University Medical Center Groningen (UMCG) between November 2008 and March 2011, had a functioning graft for at least 1 year after transplantation, were free of drug or alcohol dependence or systemic disease, and were invited to participate in the cohort. During the recruitment period, 817 KTRs were recruited, of whom 707 (87%) agreed to participate and provided written informed consent.

[0697] Example 1-2 Determined clinical performance parameters Hazard ratios and 95% confidence intervals calculated based on Cox proportional hazards regression analysis are used to evaluate the ability of uPTM-FetA in assessing the risk of renal graft failure, defined as the need for retransplantation or resumption of dialysis.

[0698] Statistics and subject counts The number of subjects required was estimated based on the following assumptions: 1. The hazard ratio (θ) of the high-risk group relative to the low-risk group is expected to be greater than 1, and this study sets the hazard ratio at 2.5. 2. According to literature statistics, approximately 20% of KTRs develop graft dysfunction. 6 Therefore, this study estimated the incidence to be 20% (pE=0.20). 3. The ratio of subjects in the low-risk group and the high-risk group is 3:1 (pL=0.75, pH=0.25). 4. The type I error (α) is 0.05. 5. Power (1-β) is 80%.

[0699] The number of samples is calculated as 7((z1-α / 2+z1-β) / (lnθ-ln(1)))2 / (pLpHpE). Under the above assumptions, 249 patients are required.

[0700] Examples 1 to 3 Data and results management Excel files containing the complete clinical records of KTRs in the TransplantLines Food and Nutrition Biobank and Cohort Study will be manually downloaded by the principal investigator and stored in a specific folder on a computer with an encrypted disk and other privacy and security measures, consistent with GDPR. The folder will be accessible only to the PI and statistician. Details regarding data storage, backup, selection, and retention are available below. Urine samples will be transferred to BPM and then stored at -80°C according to BPM's sample management procedures and will not be removed until use. After testing, test results will be recorded in an Excel file by BPM testing laboratory staff and stored in a specific folder, as described above.

[0701] Analysis of data and results This study appears to focus on determining the optimal threshold for distinguishing high- and low-risk subjects for renal graft failure in KTRs using uPTM-FetA / UCr. The primary endpoint of this study was graft failure censored at death, defined as the need for retransplantation or resumption of dialysis. The study design divided KTRs with spot urine samples into two subsets: 50% of KTRs and 50% of KTRs with Youden index. 8 This involves splitting the KTRs into a primary set, which is used to determine clinical cutoffs using a criterion, and a validation set, which consists of the remaining 50% of the KTRs and is used to validate the cutoffs.

[0702] In the routine practice of urine testing, spot urine is preferred over 24-hour urine samples, especially when 24-hour urine samples are difficult to obtain or unavailable. However, 24-hour urine tests are still commonly used for metabolic assessment of some types of kidney disease. In this study, to verify the generality of the kit's prognostic ability across different types of urine samples, we defined the KTR with uPTM-FetA and urinary creatinine measured from 24-hour urine samples as the "24-hour set" and performed a cutoff validation analysis.

[0703] After determining the cutoff in the primary set, KTRs were divided into two groups based on their uPTM-FetA / UCr levels: low-risk (uPTM-FetA / UCr < cutoff) and high-risk (uPTM-FetA / UCr ≥ cutoff). The Cox proportional hazards model and Kaplan-Meier graph / log-rank test were used to evaluate the hazard ratios and compare survival functions between the two groups. The prognostic ability of uPTM-FetA / UCr in renal graft failure was evaluated using the Cox proportional hazards model and Kaplan-Meier graph / log-rank test in both sets.

[0704] Proteinuria is commonly used as a prognostic marker after kidney transplantation 9、10It is generally accepted that proteinuria of greater than 0.5 gm per day one year after transplantation predicts the development of chronic allograft nephropathy and poor graft outcome. 11-13 To demonstrate the ability of uPTM-FetA / UCr in providing additional interpretive information in clinical use, a comparative analysis was performed to evaluate the event rate of renal graft failure compared with uPTM-FetA / cr (above and below the clinical cutoff) and proteinuria (above and below 0.5 gm per day) by using Cox regression models after adjusting for baseline risk factors of age and sex.

[0705] Examples 1 to 4 result Of the 707 KTRs, 173 and 72 patients lacked sufficient spot and 24-hour urine volumes, respectively, for uPTM-FetA measurement, and these patients were excluded, leaving 534 KTRs for spot urine-based analyses and 635 KTRs for 24-hour urine sample-based analyses.

[0706] The 534 KTRs with spot urine samples were divided into two sets: the primary set, which included 267 (50%) KTRs, and the validation set, which included 267 (50%) KTRs. The 635 KTRs with uPTM-FetA and urinary creatinine measured from 24-hour urine samples were defined as the 24-hour set.

[0707] Primary set results The mean age of the 267 KTRs was 53.93 years, 116 (43%) were women, and the eGFR was 51.69 ml / min / 1.73 m 2The median baseline protein excretion was 0.21 g / day. Of the 267 KTRs, 35 (13%) experienced renal graft failure during follow-up, with a median survival of 6.04 years (IQR, 4.62-6.40 years). The clinical cutoff for uPTM-FetA / UCr obtained by the Youden index method was 29.1 ng / mg (equivalent to an IVD103 cutoff of 1.464). According to this clinical cutoff, subjects were divided into low-risk (uPTM-FetA / UCr < 29.1 ng / mg) and high-risk (uPTM-FetA / UCr ≥ 29.1 ng / mg) groups (Table 1). From Table 1, there was a significant difference in the frequency of renal graft failure between the low-risk and high-risk groups (8.1% and 31%, p-value<0.001), and the survival time of the high-risk group was significantly shorter than that of the low-risk group (4.64 [2.82, 6.15] vs. 6.08 [5.65, 6.46], p-value<0.001). Table 1. Summary of 267 KTRs in the primary set [Table 1] JPEG2026504161000003.jpg32159Table 2. Cumulative probability (with 95% confidence interval) of the event of renal graft failure at various time points for the primary set of KTRs. [Table 2] Table 3. Cox proportional hazards regression model for the primary set [Table 3]

[0708] Notation: The reference category for uPTM-FetA / UCr is low levels of uPTM-FetA / UCr<29.1ng / mg. The reference group for gender is male. Age and eGFR are included as continuous variables.

[0709] Figure 6 shows Kaplan-Meier survival curves for KTRs with graft failure censored at death in the primary set, in some embodiments. Referring to Figure 1, Kaplan-Meier estimates indicated that KTRs in the high-risk group had a significant risk of events compared with KTRs in the low-risk group, with a log-rank p-value of <0.0001. The cumulative probability of events in the primary set by baseline uPTM-FetA / UCr risk classification at 2-5 years post-baseline is also displayed in Table 2.

[0710] Further investigation using univariate Cox proportional hazards models showed that the high-risk group had a significantly higher risk of graft failure than the low-risk group, with a hazard ratio of 4.98 (95% CI: 2.56–9.69, p <0.001). Therefore, uPTM-FetA / UCr was a significant predictor of increased risk of renal graft failure (Table 3). Multivariate Cox proportional hazards models adjusted for age, sex, and eGFR are also shown in Table 3. Although uPTM-FetA / UCr was attenuated from the univariate analysis, it remained a highly significant and independent predictor of renal graft failure risk with a hazard ratio of 2.88 (95% CI: 1.42–5.84, p = 0.003). The reduced risk likely reflects that uPTM-FetA / UCr may be associated with clinical factors, but it clearly provides additional information.

[0711] Additionally, a comparative analysis of uPTM-FetA / UCr, proteinuria, and event rates using urine samples from the primary set was provided to provide additional interpretive information for the clinical use of uPTM-FetA / UCr (Table 4). The analysis evaluated the primary endpoint of event rate compared with uPTM-FetA / UCr (above and below the cutoff of 29.1 ng / mg) and proteinuria (above and below total protein excretion of 0.5 g / day). Compared with the reference group of uPTM-FetA / UCr<29.1ng / mg and non-proteinuria, the hazard ratios were 3.01 (95% CI: 0.79-11.5, p-value=0.107), 8.82 (95% CI: 3.39-22.9, p-value<0.001), and 18.5 (95% CI: 7.77-44.2, p-value<0.001) for the second, third, and fourth categories, respectively.

[0712] Figure 2A shows Kaplan-Meier curves of KTRs in the primary set with non-proteinuria, according to a non-limiting embodiment. Figure 2B shows Kaplan-Meier curves of KTRs in the primary set with proteinuria, classified by uPTM-FetA / UCr risk category, according to a non-limiting embodiment. Referring to Figures 2A and 2B, the Kaplan-Meier curves in Figures 2A and 7BN indicated that KTRs with uPTM-FetA / UCr ≥ 29.1 ng / mg had a marginally significant risk of events compared to the low uPTM-FetA / UCr group for both non-proteinuria and proteinuria, with log-rank p-values ​​of 0.12 and 0.074, respectively. This suggests that uPTM-FetA / UCr can provide additional information to proteinuria when assessing the risk of renal graft failure. Table 4. Hazard ratios for renal graft failure by uPTM-FetA / UCr and proteinuria classification for 267 KTRs in the primary set. [Table 4]

[0713] Validation set results The mean age of the 267 KTRs was 52.32 years, 111 (42%) were women, the eGFR was 51.95 ml / min / 1.73 m2, and the median baseline protein excretion was 0.18 g / day. Of the 267 KTRs, 29 (11%) experienced renal graft failure during follow-up, and their median survival was 5.06 years (IQR, 4.47-5.36 years). According to the clinical cutoff of 29.1 ng / mg derived from the primary set, KTRs were divided into low-risk (uPTM-FetA / UCr < 29.1 ng / mg) and high-risk (uPTM-FetA / UCr ≥ 29.1 ng / mg) groups (Table 5). From Table 5, there was a significant difference in the frequency of renal graft failure between the low-risk and high-risk groups (7.6% and 29%, p-value < 0.001), and the survival time of the high-risk group was slightly significantly shorter than that of the low-risk group (4.98 [2.34, 5.34] vs. 5.06 [4.55, 5.36], p-value = 0.100). Table 5. Summary of KTRs for 267 people in the validation set [Table 5] JPEG2026504161000008.jpg32159

[0714] Figure 3 shows Kaplan-Meier survival curves for KTRs with graft failure censored at death in the validation set, according to a non-limiting embodiment. Referring to Figure 3, Kaplan-Meier estimates indicated that KTRs in the high-risk group had a significant risk of events compared with KTRs in the low-risk group, with a log-rank p-value of <0.0001. The cumulative probability of events in the validation set by baseline uPTM-FetA / UCr risk classification at 2-5 years post-baseline is also displayed in Table 6. Table 6. Cumulative probability (with 95% confidence interval) of the event of renal graft failure at various time points for subjects in the validation set. [Table 6] Table 7. Cox proportional hazards regression model for the validation set [Table 7] Notation: The reference category for uPTM-FetA / UCr is low levels of uPTM-FetA / UCr < (29.1 ng / mg). The reference group for gender is male. Age and eGFR are included as continuous variables.

[0715] Further investigation using univariate Cox proportional hazards models showed that the high-risk group had a significantly higher risk of graft failure than the low-risk group, with a hazard ratio of 4.51 (95% CI: 2.15–9.45, p <0.001). Therefore, uPTM-FetA / UCr was a significant predictor of increased risk of renal graft failure (Table 7). Multivariate Cox proportional hazards models adjusted for age, sex, and eGFR are also shown in Table 7. Although uPTM-FetA / UCr was attenuated from the univariate analysis, it remained a significant and independent predictor of renal graft failure risk with a hazard ratio of 2.36 (95% CI: 1.09–5.15, p = 0.030). The reduced risk likely reflects that uPTM-FetA / UCr may be associated with clinical factors, but it clearly provides additional information.

[0716] Additionally, a comparative analysis of uPTM-FetA / UCr, proteinuria, and event rates using urine samples from the primary set was provided to provide additional interpretive information for the clinical use of uPTM-FetA / UCr (Table 8). The analysis evaluated the primary endpoint of event rate compared with uPTM-FetA / UCr (above and below the cutoff of 29.1 ng / mg) and proteinuria (above and below total protein excretion of 0.5 g / d). Compared with the reference group of uPTM-FetA / UCr<29.1ng / mg and non-proteinuria, the hazard ratios were 2.93 (95% CI: 0.93-9.23, p-value=0.066), 3.46 (95% CI: 1.19-10.1, p-value=0.023), and 13.1 (95% CI: 5.08-33.5, p-value<0.001) for the second, third, and fourth categories, respectively.

[0717] Figure 4A shows Kaplan-Meier curves of KTRs in the validation set with non-proteinuria by uPTM-FetA / UCr risk classification, according to a non-limiting embodiment. Figure 4B shows Kaplan-Meier curves of KTRs in the validation set with proteinuria by uPTM-FetA / UCr risk classification, according to a non-limiting embodiment. Referring to Figures 4A and 4B, the Kaplan-Meier curves showed that subjects with uPTM-FetA / UCr ≥ 29.1 ng / mg had a significant event risk compared to the low uPTM-FetA / UCr group for both non-proteinuria and proteinuria, with log-rank p-values ​​of 0.061 and 0.015, respectively. This suggests that uPTM-FetA / UCr can provide additional information to proteinuria when assessing the risk of renal graft failure.

[0718] Analysis with the validation set provided positive validation and confirmed the utility of the clinical cutoff of uPTM-FetA / UCr with good predictive power for KTRs at risk for graft failure. Table 8. Hazard ratios for renal graft failure by uPTM-FetA / UCr and proteinuria classification for 267 KTRs in the validation set. [Table 8]

[0719] 24-hour set results The mean age of the 635 KTRs was 53 years, 268 (42%) were women, the mean baseline eGFR was 52.31 ml / min / 1.73 m2, and the median mean baseline protein excretion was 0.20 g / day. Of the 635 KTRs, 75 (12%) experienced renal graft failure during follow-up, and their median survival time was 5.32 years (IQR, 4.50-6.03 years). According to the clinical cutoff of 29.1 ng / mg derived from the primary set, subjects were divided into low-risk (uPTM-FetA / UCr < 29.1 ng / mg) and high-risk (uPTM-FetA / UCr ≥ 29.1 ng / mg) groups (Table 9). From Table 9, there was a significant difference in the frequency of renal graft failure between the low-risk and high-risk groups (4.8% and 19%, p-value<0.001), and the survival time of the high-risk group was significantly shorter than that of the low-risk group (5.10 [3.71, 5.80] vs. 5.44 [4.93, 6.26], p-value<0.001). Table 9. Summary of 635 KTRs in the 24-hour set [Table 9] JPEG2026504161000013.jpg34159

[0720] Figure 5 shows Kaplan-Meier survival curves for 635 KTRs with graft failure censored at death, according to non-limiting embodiments. Referring to Figure 5, Kaplan-Meier estimates showed that KTRs in the high-risk group had a significant risk of events compared with KTRs in the low-risk group, with a log-rank p-value of <0.0001. The cumulative probability of events in 24-hour sets by baseline uPTM-FetA / UCr risk classification at 2-5 years post-baseline is also displayed in Table 10. Table 10. Cumulative probability (with 95% confidence interval) of the event of renal graft failure at various time points for 635 KTRs. [Table 10] Table 11. Cox proportional hazards regression model for the 24-hour set [Table 11] Notation: The reference category for uPTM-FetA / UCr is low levels of uPTM-FetA / UCr < (29.1 ng / mg). The reference group for gender is male. Age and eGFR are included as continuous variables.

[0721] Further investigation using univariate Cox proportional hazards models showed that the high-risk group had a significantly higher risk of graft failure than the low-risk group, with a hazard ratio of 4.71 (95% CI: 2.71–8.19, p <0.001). Therefore, uPTM-FetA / UCr was a significant predictor of increased risk of renal graft failure (Table 11). Multivariate Cox proportional hazards models adjusted for age, sex, and eGFR are also shown in Table 11. Although uPTM-FetA / UCr was attenuated from the univariate analysis, it remained a highly significant and independent predictor of risk of renal graft failure with a hazard ratio of 2.80 (95% CI: 1.58–4.94, p <0.001). The reduced risk likely reflects that uPTM-FetA / UCr may be associated with clinical factors, but it clearly provides additional information.

[0722] Additionally, a comparative analysis of uPTM-FetA / UCr, proteinuria, and event rates using urine samples from the 24-hour set was provided to provide additional interpretive information for the clinical use of uPTM-FetA / UCr (Table 12). The analysis evaluated the primary endpoint of event rate compared with uPTM-FetA / UCr (above and below the cutoff of 29.1 ng / mg) and proteinuria (above and below total protein excretion of 0.5 g / d). Compared with the reference group of uPTM-FetA / UCr<29.1ng / mg and non-proteinuria, the hazard ratios were 3.08 (95% CI: 1.48-6.40, p-value=0.003), 6.10 (95% CI: 2.10-17.7, p-value<0.001), and 15.5 (95% CI: 7.90-30.5, p-value<0.001) for the second, third, and fourth categories, respectively.

[0723] Figure 6A shows Kaplan-Meier curves of KTRs in the 24-hour set with (a) no proteinuria, by uPTM-FetA / UCr risk classification, according to a non-limiting embodiment. Figure 6B shows Kaplan-Meier curves of KTRs in the 24-hour set with (b) proteinuria, by uPTM-FetA / UCr risk classification, according to a non-limiting embodiment. Referring to Figures 6A and 6B, the Kaplan-Meier curves in Figures 6A and 6B also showed that KTRs with uPTM-FetA / UCr ≥ 29.1 ng / mg had a significant event risk compared to the low uPTM-FetA / UCr group for both non-proteinuria and proteinuria, with log-rank p-values ​​of 0.013 and 0.031, respectively. This suggests that uPTM-FetA / UCr can provide additional information to proteinuria when assessing the risk of renal graft failure. Table 12. Hazard ratios for renal graft failure by uPTM-FetA / UCr and proteinuria classification for 635 KTRs in the 24-hour set. [Table 12]

[0724] conclusion The results of this study demonstrate that uPTM-FetA / UCr can effectively predict the risk of graft failure in KTR patients, regardless of age, sex, or eGFR. The prognostic value of uPTM-FetA / UCr in addition to proteinuria is clear. The clinical effectiveness of the uPTM-FetA / UCr clinical cutoff (=29.1 ng / mg) was verified in an independent validation set, and also in a 24-hour set that included uPTM-FetA and urinary creatinine measured using 24-hour urine samples.

[0725] These results indicate that uPTM-FetA / UCr can effectively predict the actual decline in eGFR. High-risk patients can receive early treatment or relevant adjustments to achieve preventive effects, which has great clinical benefits and is beneficial for KTR.

[0726] Examples 1 to 5 Additional marker combination models were tested and analyzed for incorporation into kidney transplant follow-up. A total of four models were tested and analyzed to predict graft failure in KTR: Model 1: uPTM-FetA / UCr only: Model 2: Adjusted for age, sex, and time since transplantation at enrollment (log2), as well as UACR (urinary albumin-to-creatinine ratio). Model 3 was further adjusted for eGFR (estimated glomerular filtration rate based on the creatinine and cystatin C-based CKD-EPI equation). Model 4 was further adjusted for the presence of human leukocyte antigens. Table 13. Logistic regression for graft failure over 2 years (spot urine) [Table 13]

[0727] FIG. 7 shows the receiver operating characteristic (ROC) curve for uPTM-FetA / UCr and the corresponding Kaplan-Meier curve for the marker combo model of KTRs with graft failure over 2 years using spot urine, according to non-limiting embodiments.

[0728] FIG. 8 shows receiver operating characteristic (ROC) curves and corresponding Kaplan-Meier curves of marker combo models for the combination of uPTM-FetA / UCr, age, sex, time since transplant at enrollment (log2), and UACR for KTRs with graft failure over a 2-year period using spot urine, according to non-limiting embodiments.

[0729] FIG. 9 shows receiver operating characteristic (ROC) curves for the combination of uPTM-FetA / UCr, age, sex, time since transplant at enrollment (log2), UACR, and eGFR, and the corresponding Kaplan-Meier curves of the marker combo model for KTRs with 2-year graft failure using spot urine, according to non-limiting embodiments.

[0730] FIG. 10 shows receiver operating characteristic (ROC) curves of uPTM-FetA / UCr, age, sex, time since transplant at enrollment (log2), UACR, eGFR, and presence of human leukocyte antigen class II antibodies (HLA II) and corresponding Kaplan-Meier curves of marker combo models for KTRs with 2-year graft failure using spot urine, according to non-limiting embodiments.

[0731] Referring to Table 13 and Figures 7-10, the combination of additional markers resulted in increased AUC values ​​for KTRs with graft failure at 2 years. Table 14. Logistic regression for graft failure over 5 years (spot urine) [Table 14]

[0732] FIG. 11 shows the receiver operating characteristic (ROC) curve for uPTM-FetA / UCr and the corresponding Kaplan-Meier curve for the marker combo model of KTRs with 5-year graft failure using spot urine, according to non-limiting embodiments.

[0733] FIG. 12 shows receiver operating characteristic (ROC) curves and corresponding Kaplan-Meier curves of marker combo models for the combination of uPTM-FetA / UCr, age, sex, time since transplant at enrollment (log2), and UACR for KTRs with graft failure over a 5-year period using spot urine, according to non-limiting embodiments.

[0734] FIG. 13 shows receiver operating characteristic (ROC) curves for the combination of uPTM-FetA / UCr, age, sex, time since transplant at enrollment (log2), UACR, and eGFR, and the corresponding Kaplan-Meier curves of the marker combo model for KTRs with 5-year graft failure using spot urine, according to non-limiting embodiments.

[0735] FIG. 14 shows receiver operating characteristic (ROC) curves of uPTM-FetA / UCr, age, sex, time since transplant at enrollment (log2), UACR, eGFR, and presence of human leukocyte antigen class II antibodies (HLA II) and corresponding Kaplan-Meier curves of marker combo models for KTRs with 5-year graft failure using spot urine, according to non-limiting embodiments.

[0736] Referring to Table 14 and Figures 11-14, the combination of additional markers resulted in increased AUC values ​​for KTR with graft failure at 5 years.

[0737] Intended purpose The Human uPTM3-DKD ELISA Kit is a colorimetric immunoassay for quantifying specific fetuin A with a specific post-translational modification (hereafter referred to as E103) in urine. This product is a non-automated IVD for prognostic use and must be performed in a competent clinical laboratory by a certified medical professional, such as a medical technician. E103 concentrations must be corrected for urinary creatinine before clinical application. The Human uPTM3-DKD ELISA Kit is used in conjunction with clinical evaluation as an aid in assessing the prognosis of renal function in kidney transplant recipients.

[0738] Technical and functional features The Human uPTM3-DKD ELISA is a competitive immunoassay. In this assay, calibrator or unknown urine samples are mixed with a monoclonal antibody (mAb) against fetuin A bearing a unique PTM and then incubated in a microplate precoated with the unique PTM fetuin A. The monoclonal antibody recognizes the unique PTM fetuin A in the calibrator or unknown sample in competition with the microplate well. After incubation, a horseradish peroxide (HRP)-conjugated secondary antibody is added, followed by incubation with 3,3',5,5'-tetramethylbenzidine (TMB) substrate. Their relative reactivities are determined by absorbance measurement at 450 nm and plotted against a predetermined unique PTM fetuin A calibration curve.

[0739] identification Table 15. Identification [Table 15]

[0740] Example 2-1 The publication, "Urinary Post-Translationally Modified Fetuin-A Protein Is Associated with Increased Risk of Graft Failure in Kidney Transplant Recipients," by Firas F. Alkaff, et. al., published in Am J Nephrol 2023 and available online at https: / / doi.org / 10.1159 / 000534829, is incorporated herein by reference in its entirety. The purpose of the clinical performance studies presented in Examples 2-1 through 2-5 was to evaluate the prognostic performance of the Human uPTM3-DKD ELISA test for graft failure in kidney transplant recipients (KTRs). In Examples 2-1 through 2-5, KT data were obtained from 24-hour urine.

[0741] Design and study population This prospective cohort study used data from the TransplantLines Food and Nutrition Biobank and Cohort Study (NCT02811835). All adult KTRs who visited the outpatient clinic at the University Medical Center Groningen (UMCG) between November 2008 and March 2011, had a functioning graft for at least 1 year after transplantation, and were free of drug or alcohol dependence or systemic disease, were invited to participate in the cohort. During the recruitment period, 817 KTRs were recruited, of whom 707 (87%) agreed to participate and provided written informed consent. For this study, participants with missing 24-hour uPTM-FetA excretion measurements were excluded from the analysis. This study was conducted in accordance with the Declaration of Helsinki and Istanbul, and the study protocol was approved by the Institutional Review Board of the UMCG (METc 2008 / 186).

[0742] The primary endpoint of this study was graft failure, defined as the need for retransplantation or (re)initiation of dialysis. Secondary endpoints were graft deterioration (graft failure or doubling of serum creatinine) and all-cause mortality. For the endpoints of graft failure and graft deterioration, KTRs who died with a functioning graft were censored at the time of death. Endpoints were recorded until September 2015. No KTRs were lost to follow-up thanks to a continuous monitoring system at the UMCG outpatient clinic.

[0743] Example 2-2 Clinical parameters All baseline measurements were performed during morning outpatient clinic visits. Blood pressure was measured twice, 15 minutes apart, and the results were averaged. Measurements were performed using a semi-automated device (Di-namap 1846, Critikon, Tampa, USA). Weight and height were measured while participants were wearing indoor clothing without shoes. Body mass index was calculated as height in meters squared (kg / m 2) and body surface area is calculated as body weight in kilograms divided by square meters (m ) using the DuBois and DuBois formula. 2 ) was estimated. Diabetes was defined according to the American Diabetes Association criteria. Estimated glomerular filtration rate (eGFR) was calculated using the creatinine-based Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) formula. Primary CMV infection was defined as CMV infection occurring in a previously seronegative KTR before transplantation of a kidney from a seropositive donor. Secondary CMV infection was defined as CMV infection occurring in a KTR who was seropositive before transplantation. Relevant donor, recipient, and transplant information was extracted from medical records as previously described.

[0744] Example 2-3 Laboratory method and uPTM-FetA measurement Blood samples were collected in the outpatient clinic in the morning after a drug-free overnight fasting period (approximately 8–12 h). For urine collection, all participants were instructed to collect a 24-h urine sample the day before the visit.

[0745] uPTM-FetA was measured using a novel Human uPTM3-DKD ELISA kit (CE IVD Marked, Bio Preventive Medicine Corp., Hsinchu, Taiwan, trade name: DNite-IVD103). This assay detects post-translationally modified fetuin A protein containing the binding peptide. Total urinary protein excretion was measured using the Biuret reaction (MEGA AU 150, Merck Diagnostica, Darmstadt, Germany). Other biochemical parameters, including serum creatinine and high-sensitivity C-reactive protein (hs-CRP), were measured using routine laboratory methods.

[0746] (statistical analysis) All data were analyzed using SPSS version 28.0 (IBMRP., Armonk, NY, USA) and R version 4.0.5 (R Foundation for Statistical Computing, Vienna, Austria). For all analyses, a p-value of <0.05 was considered statistically significant. The distribution of continuous variables was assessed by visual inspection of histograms and quantile-quantile plots. Normally distributed variables were presented as mean ± standard deviation, skewed variables as median (interquartile range), and categorical variables as frequency (valid percentage). Univariate linear regression analysis was performed to evaluate the association between 24-h uPTM-FetA excretion and clinical and biochemical parameters. After univariate analysis, 24-h uPTM-FetA excretion was adjusted for sex, serum creatinine, and 24-h urinary protein excretion ≥0.5 g / 24 h. During linear regression analysis, skewed variables were log2 transformed to meet the assumptions of linear regression. If log2 transformation failed to meet the assumptions, variables were reclassified into categorical variables according to the median or acceptable cutoff level.

[0747] Kaplan-Meier curves were used to visualize differences between graft and patient survival among KTR subgroups according to median levels of 24-h uPTM-FetA excretion (<34 μg / 24 h vs. ≥34 μg / 24 h). The significance of differences between subgroups was calculated using the log-rank test. Cox proportional hazards regression analysis was used to evaluate the association between 24-h uPTM-FetA excretion and graft failure, graft function decline, and all-cause mortality. The associations were adjusted for potential confounders. Variables associated with 24-h uPTM-FetA excretion in unadjusted linear regression analysis were considered potential confounders. In model 1, 24-h uPTM-FetA excretion was adjusted for age, sex, and time since transplantation (log2) at enrollment. In model 2, further adjustment was made for eGFR. In model 3, we further adjusted for 24-hour urinary protein excretion (log2). In model 4, we further adjusted for KTR clinical characteristics (systolic blood pressure, body surface area, smoking status, and CMV infection status) and use of growth inhibitors. In model 5, we further adjusted for donor characteristics (donor type, donor age, and donor sex). In all models (model 6), we further adjusted for hs-CRP (log2).

[0748] Schoenfeldt residuals as a function of time were performed to assess the proportional hazards assumption in the final Cox regression model for each endpoint. The proportional hazards assumption was violated for graft failure and graft decline (p = 0.004 and p = 0.005, respectively), but not for all-cause mortality (p = 0.073). Therefore, Cox models including time-dependent covariates were used to calculate hazard ratios (HRs) over time for graft failure and graft decline. HRs are presented as a function of doubling of 24-hour uPTM-FetA excretion, along with 95% confidence intervals (95% CI).

[0749] For the primary endpoint, we assessed potential interactions between age, sex, eGFR, and 24-hour urinary protein excretion and 24-hour uPTM-FetA excretion by adding interaction terms to the full model. For sensitivity analyses, we reassessed the association between 24-hour uPTM-FetA excretion and graft failure after excluding outliers. In this study, outliers were defined as values ​​that deviated more than two standard deviations from the mean log2 24-hour uPTM-FetA excretion. Subsequently, we used uPTM-FetA concentrations and uPTM-FetA concentrations indexed for creatinine (uPTM-FetA / creatinine ratio) instead of 24-hour uPTM-FetA excretion.

[0750] For all cross-sectional analyses, the original unimputed dataset was used, and variables with >20 missing values ​​(3.16%) were reported in table footnotes. For all prospective analyses, multiple imputation using fully conditional specification was performed using the R package "mouse" to account for missing data for variables other than 24-h uPTM-FetA excretion (number of multiple imputations = 10).

[0751] Examples 2-4 result Figure 15 shows a flowchart of the selection of the study population, according to a non-limiting embodiment. Referring to Figure 15, a total of 627 KTRs were included in the study. The mean age was 53 ± 13 years, 42% were female, the median time since transplant was 5.3 (1.9-12.2) years, and the mean eGFR was 52 ± 20 mL / min / 1.73 m 2The median 24-hour uPTM-FetA excretion was 34 (17-74) μg / 24 hours. The median 24-hour urinary protein excretion was 0.21 (0.01-0.34) g / 24 hours, with 504 patients (81%) having a urinary protein excretion of <0.5 g / 24 hours and only 8 patients (1%) having a urinary protein excretion of >3 g / 24 hours. More detailed baseline characteristics of the study population are presented in Table 2-1. Referring to Table 2-1, normally distributed variables are presented as mean ± standard deviation, skewed variables are presented as median (interquartile range), and categorical variables are presented as number (valid percentage). Smoking status was missing in 39 patients, alcohol consumption status was missing in 66 patients, CMV infection status was missing in 52 patients, hemoglobin A1C was missing in 21 patients, and hs-CRP level was missing in 32 patients. All other variables had missing values ​​in <20 patients. eGFR, estimated glomerular filtration rate calculated using the creatinine-based CKD-EPI equation; HLA, human leukocyte antigen; hs-CRP, high-sensitivity C-reactive protein; mTOR, mammalian target of rapamycin; normalized beta, standardized beta coefficient; uPTM-FetA, urinary posttranslationally modified fetuin A; BMI, body mass index. Variables were log2-transformed to meet the assumptions in linear regression analysis. Table 2-1. Baseline characteristics and linear regression analysis of 24-hour uPTM-FetA excretion [Table 16]

[0752] Figure 16 shows a scatter plot and visual representation of the correlation between 24-hour uPTM-FetA excretion and serum creatine levels, according to a non-limiting embodiment. Figure 17 shows a scatter plot and visual representation of the correlation between 24-hour uPTM-FetA excretion and eGFR, according to a non-limiting embodiment. Figure 18 shows a scatter plot and visual representation of the correlation between 24-hour uPTM-FetA excretion and 24-hour albumin excretion, according to a non-limiting embodiment. Figure 19 shows a scatter plot and visual representation of the correlation between 24-hour uPTM-FetA excretion and 24-hour urinary protein, according to a non-limiting embodiment. Referring to Figures 16-19, 24-hour uPTM-FetA excretion was negatively correlated with eGFR and positively correlated with plasma creatinine concentration, urinary albumin, and 24-hour urinary protein excretion.

[0753] Cross-sectional association of 24-hour uPTM-FetA excretion with clinical and biochemical parameters In univariate linear regression analysis, 24-h uPTM-FetA excretion was most strongly associated with 24-h urinary protein excretion of ≥ 0.5 g / 24 h (standardized beta coefficient [Std.β] 0.94, p < 0.001). Furthermore, 24-h uPTM-FetA excretion was also significantly associated with 24-h urinary albumin excretion (Std.β 0.45, p < 0.001), serum creatinine (Std.β 0.34, p < 0.001), and eGFR (Std.β -0.27, p < 0.001). Furthermore, female gender was also significantly associated with higher 24-h uPTM-FetA excretion (Std.β -0.22, p = 0.006). Older donors, living donors, use of growth inhibitors, and 24-hour urinary albumin excretion were significantly associated with increased 24-hour uPTM-FetA excretion, serum creatinine, and 24-hour urinary protein excretion ≥ 0.5 g / 24 hours, regardless of gender. In contrast, longer post-transplant time and congenital primary renal disease were negatively associated with 24-hour uPTM-FetA excretion.

[0754] Prospective analysis of the association between 24-hour uPTM-FetA excretion and graft failureTable 2-2 shows a prospective analysis of the association of 24-hour uPTM-FetA excretion with death-censored graft failure in 627 KTRs. Referring to Table 2-2, Cox proportional hazards regression analysis was performed to evaluate the association between 24-hour uPTM-FetA excretion and the risk of death-censored graft failure (i.e., the need for retransplantation or (re)initiation of dialysis). Model 1 was adjusted for age, sex, and time since transplantation at inclusion (log2). Model 2 was further adjusted for eGFR based on the creatinine-based CKD-EPI equation. Model 3 was further adjusted for 24-hour urinary protein excretion (log2). Model 4 was further adjusted for KTR clinical characteristics (systolic blood pressure, body surface area, smoking status, and CMV infection status) and use of growth inhibitors. Model 5 was further adjusted for donor characteristics (donor type, donor age, and donor sex). Model 6 was further adjusted for serum high-sensitivity C-reactive protein (log2), 95% CI, 95% confidence interval, HR, hazard ratio, uPTM-FetA, and urinary post-translational modified fetuin A. Figure 20 shows Kaplan-Meier curves for graft failure censored at death below and above the median 24-hour urinary post-translational modified fetuin A excretion level, according to non-limiting embodiments. Referring to Table 2-2 and Figure 20, during a median follow-up of 5.3 years (range, 4.5-6.0 years) after baseline measurements, 73 (12%) KTRs developed graft failure. The most frequent causes of graft failure were chronic graft dysfunction (77%) and recurrence of primary renal disease (10%). Other causes of graft failure include vascular problems and infection. In KTRs with baseline 24-hour uPTM-FetA excretion levels below and above the median, the rates of graft failure were 4% and 19%, respectively (plog rank < 0.001). The association between 24-hour uPTM-FetA excretion and risk of graft failure was not consistent over time (Schoenfeld residual = 0.004). 24-hour uPTM-FetA excretion was associated with graft failure within 1 year or 1 to 3 years after baseline measurement in unadjusted models.However, the association was lost after adjustment for confounders (HR 0.99, 95% CI: 0.77-1.27 and 0.96, 95% CI: 0.78-1.19, respectively). In contrast, 24-hour uPTM-FetA excretion was associated with an increased risk of graft failure 3 years after baseline measurement (HR 1.85, 95% CI: 1.55-2.22), and the association remained significant even after adjusting for potential confounders (HR 1.31, 95% CI: 1.06-1.61) (Table 2). Regarding the association between 24-hour uPTM-FetA excretion and graft failure, there were no interactions between 24-hour uPTM-FetA excretion and sex, age, eGFR, or 24-hour urinary protein excretion (p values ​​for all interactions >0.05). Table 2. Prospective analysis of the association of 24-hour uPTM-FetA excretion with death-censored graft failure in 627 KTRs [Table 17]

[0755] Sensitivity analysis Thirty-four (5.4%) KTRs with outliers were identified (11 KTRs with 24-hour uPTM-FetA excretion <3.33 μg / 24 h and 23 KTRs with uPTM-FetA excretion >408 μg / 24 h). Table 2-3 shows a prospective analysis of the association between 24-hour uPTM-FetA excretion and death-censored graft failure, excluding outliers, according to some embodiments. With reference to Table 2-3, outliers were defined as ±2 standard deviations from the mean log2-transformed 24-hour uPTM-FetA excretion (<3.33 μg / 24 h and >408 μg / 24 h). Based on this, 34 KTRs were excluded, leaving 593 KTRs for Cox proportional hazards regression analysis. Model 1 was adjusted for age, sex, and time since transplantation at enrollment (log2). Model 2 was further adjusted for estimated glomerular filtration rate based on the creatinine-based CKD-EPI equation. Model 3 was further adjusted for 24-hour urinary protein excretion (log2). Model 4 was further adjusted for KTR clinical characteristics (systolic blood pressure, body surface area, smoking status, and CMV infection status) and use of growth inhibitors. Model 5 was further adjusted for donor characteristics (donor type, donor age, and donor sex). Model 6 was further adjusted for serum high-sensitivity C-reactive protein (log2), 95% CI, 95% CI, HR, hazard ratio, uPTM-FetA, and urinary post-translational modified fetuin A. After excluding outliers, 24-hour uPTM-FetA excretion remained independently associated with the risk of graft failure only 3 years after baseline measurement (HR 1.77, 95% CI: 1.25-2.49). Table 2-3. Prospective analysis of the association between 24-hour uPTM-FetA excretion and death-censored graft failure, excluding outliers in 24-hour uPTM-FetA excretion. [Table 18]

[0756] In other sensitivity analyses, uPTM-FetA concentrations and uPTM-FetA / creatinine ratios were used instead of 24-hour uPTM-FetA excretion. Tables 2-4 show a prospective analysis of the association between uPTM-FetA concentrations and graft failure in 627 kidney transplant recipients. Referring to Tables 2-4, Cox proportional hazards regression analyses were performed to evaluate the association between uPTM-FetA excretion and the risk of graft failure censored by death. Model 1 was adjusted for age, sex, and time since transplantation at inclusion (log2). Model 2 was further adjusted for estimated glomerular filtration rate based on the creatinine-based CKD-EPI equation. Model 3 was further adjusted for 24-hour urinary protein excretion (log2). Model 4 was further adjusted for clinical characteristics of the KTR (systolic blood pressure, body surface area, smoking status, and CMV infection status) and use of growth inhibitors. Model 5 was further adjusted for donor characteristics (donor type, donor age, and donor sex). Model 6 was further adjusted for serum high-sensitivity C-reactive protein (log2), 95% CI, 95% confidence interval, HR, hazard ratio, uPTM-FetA, and urinary post-translational modified fetuin A. Tables 2-4. Prospective analysis of the association between uPTM-FetA concentrations and graft failure in 627 kidney transplant recipients. [Table 19]

[0757] Tables 2-5 show a prospective analysis of the association between the uPTM-FetA / creatinine ratio and graft failure in 627 kidney transplant recipients, according to some embodiments. Referring to Tables 2-5, Cox proportional hazards regression analysis was performed to evaluate the association between the uPTM-FetA / urinary creatinine ratio and the risk of graft failure censored by death. Model 1 was adjusted for age, sex, and time since transplantation at enrollment (log2). Model 2 was further adjusted for estimated glomerular filtration rate based on the creatinine-based CKD-EPI equation. Model 3 was further adjusted for 24-hour urinary protein excretion (log2). Model 4 was further adjusted for clinical characteristics of the KTR (systolic blood pressure, body surface area, smoking status, and CMV infection status) and use of growth inhibitors. Model 5 was further adjusted for donor characteristics (donor type, donor age, and donor sex). Model 6 was further adjusted for serum high-sensitivity C-reactive protein (log2), 95% CI, 95% confidence interval, HR, hazard ratio, uPTM-FetA, and urinary post-translationally modified fetuin A. Table 2-5. Prospective analysis of the association between the uPTM-FetA·creatinine ratio and graft failure in 627 kidney transplant recipients. [Table 20]

[0758] Referring to Tables 2-3 and 2-4, similarly, uPTM-FetA concentration and uPTM-FetA / creatinine ratio were also significantly associated with graft failure only 3 years after baseline measurement, even after adjusting for potential confounders (HR 1.35, 95% CI: 1.10-1.65, and HR 1.36, 95% CI: 1.10-1.67, respectively).

[0759] Secondary analysis of the association between 24-hour uPTM-FetA and graft function decline and all-cause mortality During a median follow-up of 5.3 years (range, 4.0-6.0 years) after baseline measurements, 121 (19%) KTRs developed graft decline. Figure 21 shows Kaplan-Meier curves for death-censored graft decline below and above the median 24-hour urinary post-translational modified fetuin A excretion level, according to a non-limiting embodiment. Tables 2-6 show Supplementary Table 5. A prospective analysis of the association between 24-hour uPTM-FetA excretion and death-censored graft decline in 627 kidney transplant recipients is shown. Referring to Tables 2-6, Cox proportional hazards regression analysis was performed to evaluate the association between 24-hour uPTM-FetA excretion and the risk of graft decline. Model 1 was adjusted for age, sex, and time since transplantation (log2) at inclusion. Model 2 was further adjusted for estimated glomerular filtration rate based on the creatinine-based CKD-EPI equation. Model 3 was further adjusted for 24-hour urinary protein excretion (log2). Model 4 was further adjusted for KTR clinical characteristics (systolic blood pressure, body surface area, smoking status, and CMV infection status) and use of growth inhibitors. Model 5 was further adjusted for donor characteristics (donor type, donor age, and donor sex). Model 6 was further adjusted for serum high-sensitivity C-reactive protein (log2), 95% CI, 95% confidence interval, HR, hazard ratio, uPTM-FetA, and urinary post-translational modification fetuin A. Referring to Tables 2-6 and Figure 21, the rates of graft function decline were 9% and 30%, respectively, in KTRs with baseline 24-hour uPTM-FetA excretion below and above the median, respectively (plog rank < 0.001). The association between 24-hour uPTM-FetA excretion and risk of graft failure was not consistent over time (Schoenfeld residual = 0.005). 24-hour uPTM-FetA excretion was significantly associated with an increased risk of graft failure (HR 1.64, 95% CI: 1.41-1.92) only 3 years after baseline measurement, and the association remained significant after adjustment for potential confounders (HR 1.39, 95% CI: 1.17-1.65). Table 2-6. Prospective analysis of the association between 24-hour uPTM-FetA excretion and graft function decline censored by death in 627 kidney transplant recipients. [Table 21]

[0760] During a median follow-up of 5.4 years (range, 4.9-6.1) after baseline measurements, 132 (21%) KTRs died. Figure 22 shows Kaplan-Meier curves for all-cause mortality among KTRs with 24-hour urinary post-translationally modified fetuin A excretion levels below and above the median, according to non-limiting embodiments. There was no significant difference in mortality (plog rank = 0.2) among KTRs with 24-hour uPTM-FetA excretion levels below (19%) and above (23%) the median level, respectively. Tables 2-7 show a prospective analysis of the association between 24-hour uPTM-FetA excretion levels and all-cause mortality in 627 kidney transplant recipients, according to non-limiting embodiments. Referring to Tables 2-7, Cox proportional hazards regression analysis was performed to evaluate the association between 24-hour uPTM-FetA excretion levels and the risk of all-cause mortality. Model 1 was adjusted for age, sex, and time since transplantation at enrollment (log2). Model 2 was further adjusted for estimated glomerular filtration rate based on the creatinine-based CKD-EPI equation. Model 3 was further adjusted for 24-hour urinary protein excretion (log2). Model 4 was further adjusted for KTR clinical characteristics (systolic blood pressure, body surface area, smoking status, and CMV infection status) and use of growth inhibitors. Model 5 was further adjusted for donor characteristics (donor type, donor age, and donor sex). Model 6 was further adjusted for serum high-sensitivity C-reactive protein (log2), 95% CI, 95% CI, HR, hazard ratio, uPTM-FetA, and urinary post-translational modification fetuin A. Unadjusted and adjusted Cox regression models did not demonstrate a positive association between 24-hour uPTM-FetA excretion and all-cause mortality. Table 2-7. Prospective analysis of the association between 24-hour uPTM-FetA excretion and all-cause mortality in 627 kidney transplant recipients. [Table 22]

[0761] Consideration In this cohort of 627 KTRs, 24-h uPTM-FetA excretion was most strongly associated with 24-h urinary protein excretion ≥ 0.5 g / 24 h. Additionally, shorter time since transplantation, living donor, older donor age, congenital primary renal disease, use of growth inhibitors, and 24-h urinary albumin excretion were independently associated with higher 24-h uPTM-FetA excretion. In a prospective analysis, 24-h uPTM-FetA excretion was independently associated with an increased risk of graft failure. However, the risk was not consistent over time, with elevated risk observed only > 3 years after baseline measurement. The association was robust across several sensitivity analyses. Similarly, 24-h uPTM-FetA excretion was associated with decreased graft function only 3 years after baseline measurement. In contrast, 24-hour uPTM-FetA excretion was not associated with an increased risk of death.

[0762] The single-chain precursor form of human fetuin-A contains an A chain, a connecting peptide, and a B chain in addition to the signaling peptide. This precursor protein is then post-translationally modified by glycosylation and proteolysis. During proteolysis, the connecting peptide region is removed from the precursor protein, and the single-chain form is converted to a mature two-chain form linked by disulfide bonds. Under certain conditions in which proteolysis does not occur, single-chain fetuin-A is instead phosphorylated, primarily at serine residues in the connecting peptide region. The ELISA kit used in this study detects only the latter post-translationally modified form of fetuin-A.

[0763] In healthy adults, the kidney does not express fetuin A. However, upon injury, proximal tubular epithelial cells (PTECs) express fetuin A and increase their ability to release fetuin A to the luminal side of the tubule. Zhou et al. demonstrated that fetuin A was predominantly present in the urinary exosomal fraction, rather than the non-exosomal fraction, in rat models of cisplatin-induced and ischemia / reperfusion-induced AKI, indicating that fetuin A is produced by PTECs. Recently, Rudloff et al. demonstrated that PTECs can locally produce fetuin A under hypoxic conditions after stimulation with hypoxia-inducible transcription factors. It has been postulated that the presence of fetuin A within the proximal tubules protects the kidney from hypoxia-induced renal inflammation by preventing macrophage shift to the proinflammatory M1 macrophage state and helps protect the kidney from hypoxia-induced fibrosis by antagonizing transforming growth factor-β signaling.

[0764] Both acute and chronic kidney injury are closely related to the development of hypoxia. In the setting of kidney transplantation, kidneys are exposed to hypoxia and various conditions that enhance hypoxic injury, such as ischemia and reperfusion, as well as postoperative vascular complications. Among all structures within the kidney, PTECs are the most vulnerable due to their high activity and oxygen demand. Because hypoxia plays a major role in the progression of kidney disease and can be present relatively early in kidney injury, even before the onset of structural damage, early identification of this condition may be beneficial to prevent further deterioration. In both cisplatin-induced and ischemia / reperfusion-induced AKI, urinary fetuin A excretion increased before the rapid rise in serum creatinine and before morphological injury was present. In this study, uPTM-FetA was found to be significantly associated with an increased risk of graft failure and graft function decline 3 years after baseline measurements. Since one of the most important requirements for a biomarker is to reflect the underlying pathophysiology of the disease, measurement of urinary uPTM-FetA may offer additional advantages as a biomarker for early detection of graft injury in KTR over currently used parameters such as serum creatinine or proteinuria.

[0765] This study has three important limitations that need to be mentioned. First, it was conducted at a single center in the Netherlands with an overrepresented Caucasian population. Second, because this was an observational study, residual confounding may still exist despite the number of potential confounders adjusted for, and the nature of this study precludes reliable conclusions regarding causality. Third, because this was a pilot study, p values ​​were not adjusted for multiple testing. Nevertheless, this was the first study to prospectively evaluate the association between urinary fetuin-A and long-term outcomes in the setting of kidney transplantation.

[0766] In conclusion, 24-hour uPTM-FetA excretion is significantly associated with decreased renal function and increased 24-hour urinary albumin and protein excretion. Prospectively, 24-hour uPTM-FetA excretion is independently associated with an increased risk of graft failure and graft function decline beyond 3 years after measurement. Our findings suggest that uPTM-FetA may be used as a clinical marker allowing early detection of graft failure in KTRs. Further studies with larger and more heterogeneous KTR populations are needed to confirm our findings. Design and Study Population

[0767] Example 3-1 Design and study population This prospective cohort study used data from the TransplantLines Food and Nutrition Biobank and Cohort Study (NCT03272841). All adult KTRs who visited the outpatient clinic at the University Medical Center Groningen (UMCG) between November 2008 and March 2011, had a functioning graft for at least 1 year after transplantation, and were free of drug or alcohol dependence or systemic disease, were invited to participate in this cohort (18). During the recruitment period, 817 KTRs were recruited, of whom 707 (87%) agreed to participate and provided written informed consent. For this study, participants lacking available 24-hour uC-FetA excretion measurements were excluded from the analysis. This study was conducted in accordance with the Declaration of Helsinki, and the study protocol was approved by the Institutional Review Board (METc 2008 / 186).

[0768] The primary endpoint of the study was graft decline (defined as a doubling of serum creatinine or graft failure, i.e., the need for retransplantation or (re)initiation of dialysis), censored by death. The secondary endpoint was all-cause mortality. Outcomes were recorded until September 2015. No participants were lost to follow-up.

[0769] Example 3-2 Clinical parameters All measurements were performed during morning outpatient clinic visits. Blood pressure and heart rate were measured three times at 15-minute intervals, and the results were averaged. Measurements were performed using a semi-automatic device (Dinamap 1846, Critikon, Tampa, USA). Weight and height were measured while participants were wearing indoor clothing without shoes. Body mass index (BMI) was calculated as height in meters squared (kg / m). 2 ) and body surface area (BSA) was calculated as body weight in kilograms divided by square meters (m ) using the DuBois and Dubois formula. 2 ) was estimated. Diabetes was defined according to the American Diabetes Association criteria. Estimated glomerular filtration rate (eGFR) was calculated using the creatinine- and cystatin C-based Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation. Relevant donor, recipient, and transplant information was extracted from medical records as previously described.

[0770] Example 3-3 Laboratory method and uC-FetA measurement Blood samples were collected in the outpatient clinic in the morning after a drug-free overnight fasting period (approximately 8–12 h). For urine collection, all participants were instructed to collect a 24-hour urine sample the day before the visit.

[0771] Cystatin C concentrations were measured in EDTA plasma using a validated particle-enhanced immunoturbidimetric assay (Gentian, Moss, Norway). A calcification tendency test to assess the maturation time (T50) of calcium protein particles was measured using a nanoparticle-based test. Total urinary protein excretion was measured using the Biuret reaction (MEGA AU 150, Merck Diagnostica, Darmstadt, Germany). Urinary L-type fatty acid binding protein (L-FABP) was measured using a human uL-FABP ELISA assay kit (CMIC Holdings Co., Tokyo, Japan). Other biochemical parameters, including creatinine, high-sensitivity C-reactive protein (hs-CRP), and lipid status, were measured using routine laboratory methods. uC-FetA was measured using a novel Human uPTM3-DKD ELISA kit (manufacturer: Bio Preventive Medicine Corp., trade name: DNite-IVD103).

[0772] All data for statistical analysis were analyzed using SPSS version 28.0 (IBMRP., Armonk, NY, USA) and R version 4.2.1 (R Foundation for Statistical Computing, Vienna, Austria). For all analyses, a p value of <0.05 was considered significant. Baseline characteristics were presented as mean ± standard deviation, median [interquartile range], and frequency (percentage) for normally distributed, skewed, and nominal data, respectively. Visual evaluation of QQ plots and histograms was performed to determine whether data were normally distributed or skewed. Univariate linear regression analysis was performed to evaluate the association between uC-FetA and clinical and biochemical parameters. After univariate analysis, uC-FetA was adjusted for sex, serum creatinine, and 24-hour urinary protein excretion. During linear regression analysis, skewed data were log2-transformed to meet the assumptions of linear regression.

[0773] The association between uC-FetA and death-censored graft decline and all-cause mortality was assessed using Kaplan-Meier curves, and the significance of intertertile differences was calculated using the log-rank test. To further evaluate the association between uC-FetA and outcomes, Cox proportional hazards regression analyses were performed, adjusting for potential confounders. In model 1, 24-hour uC-FetA excretion was adjusted for age, sex, and time since transplantation at enrollment. In model 2, eGFR was further adjusted for based on the creatinine- and cystatin C-based CKD-EPI equation. In model 3, 24-hour urinary protein excretion (log2) was further adjusted. In model 4, we further adjusted for the presence of human leukocyte antigen class II antibodies, donor type, and donor age. In model 5, we further adjusted for hs-CRP. The final model (model 6) further adjusted for the use of antiproliferative medications. Schoenfeld residues were visually confirmed and tested, and the final model did not violate the assumption of proportionality of hazards (p = 0.115 for graft decline censored at death, p = 0.580 for all-cause mortality). Potential interactions between age, sex, eGFR, and 24-hour urinary protein excretion and 24-hour uC-FetA excretion were assessed by adding interaction terms to the final model. Hazard ratios are presented with 95% confidence intervals (95% CI) for each doubling of 24-hour uC-FetA.

[0774] Because we identified an interaction between uC-FetA and eGFR, with graft function decline censored at death as the outcome, we repeated the final Cox regression model in subgroups. The median eGFR value for patients with graft decline (eGFR = 30 mL / min / 1.73 m²) was used as the cutoff to separate the two groups. For all cross-sectional analyses, the original unimputed dataset was used, and variables with >20 missing values ​​(3.16%) are reported in table footnotes. For all prospective analyses, multiple imputation using fully conditional specifications was performed (number of multiple imputations = 10) using the R package "mouse" to account for missing data among variables other than 24-hour uC-FetA excretion.

[0775] Examples 3-4 result Baseline characteristics Figure 23 shows a flowchart of study participant selection, according to a non-limiting embodiment. Referring to Figure 23, a total of 632 KTRs were included in this study. The mean age was 53 ± 13 years, 42% were female, the median time since transplant was 5.3 [1.7-12.2] years, and the mean eGFR was 45.3 ± 18.7 mL / min / 1.73. The median 24-hour uC-FetA excretion was 33.9 [17.2-74] μg / 24 hours. The median 24-hour urinary protein excretion was 0.21 [0.2-0.34] g / 24 hours, of which 511 (81%) had a urinary protein excretion of <0.5 grams / 24 hours and 8 (1.26%) had a urinary protein excretion of >3 grams / 24 hours. More detailed baseline characteristics of the study population are presented in Table 1. [Table 23] JPEG2026504161000028.jpg104159

[0776] Normally distributed data are presented as mean ± standard deviation, skewed data are presented as median [interquartile range], and categorical data are presented as number (valid percentage). Variables were log2-transformed to meet the assumptions of linear regression analysis. Diabetes was defined according to the American Diabetes Association criteria. Smoking status was missing in 39 patients, alcohol consumption status was missing in 66 patients, HbA1C was missing in 22 patients, hs-CRP level was missing in 33 patients, creatinine clearance was missing in 26 patients, and urinary L-type fatty acid-binding protein was missing in 61 patients. All other variables had missing values ​​in <20 patients. Abbreviations: eGFR, estimated glomerular filtration rate calculated using the creatinine- and cystatin C-based CKD-EPI equation; HLA, human leukocyte antigen; hs-CRP, high-sensitivity C-reactive protein; L-FABP, L-type fatty acid-binding protein; mTOR, mammalian target of rapamycin; uC-FetA, urinary binding peptide-containing fetuin A. Cross-sectional association of 24-hour uC-FetA excretion with clinical and biochemical parameters. Univariate linear regression analysis showed that male gender was significantly associated with increased 24-hour uC-FetA excretion (β = 0.11). Older donors, living donors, and use of growth inhibitors were significantly associated with increased uC-FetA excretion, serum creatinine, and 24-hour urinary protein excretion, regardless of gender. In contrast, a longer post-transplant period and higher levels of circulating aspartate aminotransaminase (AST) were negatively associated with uC-FetA excretion.

[0777] In univariate linear regression analysis, 24-h uC-FetA excretion was most strongly associated with 24-h urinary L-FABP excretion (standardized β = 0.50) and 24-h urinary protein excretion (standardized β = 0.44). uC-FetA excretion was also positively associated with plasma NGAL and plasma fibroblast growth factor 23 (FGF23) (standardized β = 0.24 and β = 0.19, respectively) and inversely associated with eGFR and T50 (standardized β = -0.29 and β = -0.17). After adjustment for sex, serum creatinine, and 24-h urinary protein excretion, urinary L-FABP remained significantly associated with uC-FetA excretion (standardized β = 0.32), whereas plasma NGAL, plasma FGF23, and T50 lost their associations (Table 1). Prospective analysis of the association between 24-hour uC-FetA excretion and death-censored graft function decline. During a median follow-up of 5.3 years (range, 4.0-6.0 years) after baseline measurement, 121 (19%) KTRs developed graft function decline. The rates of graft function decline were 8%, 16.6%, and 32.9% in KTRs in the lowest, middle, and highest tertiles of baseline 24uC-FetA excretion, respectively (plog rank <0.001) (Figure 24). Cox regression analysis showed that KTRs in tertiles 2 and 3 of 24-hour uC-FetA excretion were at higher risk of graft function decline compared with KTRs in the lowest tertile of uC-FetA excretion (HR 2.18, 95% CI: 1.22-3.90, and HR 4.79, 95% CI: 2.82-8.14, respectively). The association of tertile 3 of 24-hour uC-FetA excretion remained significant even after adjusting for potential confounders (HR 2.68, 95% CI: 1.49-4.82). Similarly, 24-hour uC-FetA excretion was also associated with a higher risk of graft function decline on a continuous scale (HR 1.19 per doubling increase, 95% CI: 1.06-1.33) even after adjusting for potential confounders (Table 2).

[0778] Table 2. Prospective analysis of the association between 24-hour uC-FetA excretion and graft function decline censored by death in 632 kidney transplant recipients. [Table 24]

[0779] Cox proportional hazards regression analysis was performed to evaluate the association between 24-hour uC-FetA excretion and the risk of graft function decline censored by death (a doubling of serum creatinine or graft failure, i.e., the need for retransplantation or (re)initiation of dialysis). Model 1 was adjusted for age, sex, and time since transplantation at inclusion (log2). Model 2 was further adjusted for estimated glomerular filtration rate based on the creatinine- and cystatin C-based CKD-EPI equation. Model 3 was further adjusted for 24-hour urinary protein excretion (log2). Model 4 was further adjusted for the presence of human leukocyte antigen class II antibodies, donor type, and donor age. Model 5 was further adjusted for serum high-sensitivity C-reactive protein (log2). Model 6 was further adjusted for the use of growth inhibitors. CI, confidence interval; HR, hazard ratio; uC-FetA; urinary binding peptide-containing fetuin A.

[0780] Examples 3-5 Sensitivity analysis There was no interaction between 24-hour uC-FetA excretion and sex, age, or 24-hour urinary protein excretion for the association between 24-hour uC-FetA excretion and graft function decline (p-values ​​for interaction = 0.07, 0.913, and 0.202, respectively). However, there was a significant interaction with eGFR (p-value for interaction = 0.01). A subgroup sensitivity analysis was performed on the final model (Model 6). The analysis showed that 24-hour uC-FetA excretion was significantly associated with graft function decline only in patients with eGFR ≥ 30 mL / min / 1.73 m² (HR 1.19, 95% CI 1.01-1.41, p = 0.040), but not in patients with eGFR < 30 mL / min / 1.73 m² (HR 1.02, 95% CI 1.02-1.41, p = 0.040), as presented in Figure 25. 0.84-1.23, p=0.858. FIG. 25 shows a forest plot of the association between uC-FetA and death-censored graft function decline in subgroups, according to non-limiting embodiments. Referring to FIG. 25, a forest plot of the association between uC-FetA and death-censored graft function decline in subgroups. The model was adjusted for age, sex, log2 of time since transplant at enrollment, eGFR, log2 of 24-hour urinary protein excretion, presence of human leukocyte antigen class II antibodies, donor type, donor age, log2 of serum high-sensitivity C-reactive protein, and the full model of growth inhibitor use.

[0781] Prospective analysis of the association between uC-FetA and all-cause mortality In a secondary analysis, we evaluated the association between uC-FetA and all-cause mortality. During a median follow-up of 5.4 years (range, 4.9–6.1 years) after baseline measurement, 133 (21%) KTRs died. There were no significant differences in mortality across uC-FetA tertiles (18.5% in the first tertile, 20.4% in the second tertile, and 24.3% in the third tertile; plog rank = 0.282) (Figure 26). Unadjusted and adjusted Cox regression models also showed no positive association between 24-hour uC-FetA excretion and all-cause mortality (Table 3). [Table 25]

[0782] Cox proportional hazards regression analysis was performed to evaluate the association between 24-hour uC-FetA excretion and risk of all-cause mortality. Model 1 was adjusted for age, sex, and time since transplantation at enrollment (log2). Model 2 was further adjusted for estimated glomerular filtration rate based on the creatinine- and cystatin C-based CKD-EPI equation. Model 3 was further adjusted for 24-hour urinary protein excretion (log2). Model 4 was further adjusted for the presence of human leukocyte antigen class II antibodies, donor type, and donor age. Model 5 was further adjusted for serum high-sensitivity C-reactive protein (log2). Model 6 was further adjusted for use of growth inhibitors. CI, confidence interval; HR, hazard ratio; uC-FetA; urinary C-peptide-containing fetuin A.

[0783] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It is understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention, and that methods and structures within the scope of these claims and their equivalents be covered thereby.

[0784] Examples 3-6 The Human uPTM3-DKD ELISA is a colorimetric immunoassay for the quantitative measurement of unique fetuin A with specific post-translational modifications (PTMs) in human urine and should be performed in a competent clinical laboratory by qualified medical professionals, such as medical technologists.

[0785] Principle of this test The Human uPTM3-DKD ELISA is a competitive immunoassay. In this assay, calibrator or unknown urine samples are mixed with a monoclonal antibody (mAb) against fetuin A bearing a unique PTM and then incubated in a microplate pre-bound with unique PTM fetuin A. The monoclonal antibody recognizes the unique PTM fetuin A in the calibrator or unknown sample in competition with the microplate well. After incubation, a horseradish peroxide (HRP)-conjugated secondary antibody is added, followed by incubation with 3,3',5,5'-tetramethylbenzidine (TMB) substrate. Their relative reactivities are determined by absorbance measurements at 450 nanometers (nm) and plotted against a predetermined unique PTM fetuin A calibration curve.

[0786] Purpose of use The Human uPTM3-DKD ELISA is a colorimetric immunoassay for the quantitative measurement of a unique fetuin A with a specific post-translational modification (PTM) in human urine. It should be performed in a competent clinical laboratory by qualified medical professionals, such as medical technicians. This product is intended for in vitro diagnostic use as an aid in the risk assessment of renal complications in diabetic patients. Furthermore, the uPTM3-DKD test may be applicable to accurately distinguish between those with progressive renal decline in type 2 diabetes accompanied by microalbuminuria. [Table 26]

[0787] Reagent preparation 1. Fetuin-A (E103) calibrator with human-specific PTMs Reconstitute the E103 calibrator with 0.2 mL of distilled or deionized water, leave at room temperature for 10 minutes, and gently mix until completely dissolved. The reconstituted E103 calibrator has a concentration of 5 μg / mL. Dilute the 5 μg / mL E103 calibrator five-fold with diluent and then gently mix to obtain a 1 μg / mL E103 calibrator. The serial dilution procedure for generating all calibrators to establish the E103 calibration curve is shown in the table below. All calibrators should be prepared and thoroughly mixed immediately before use. [Table 27]

[0788] 2.1x Wash Buffer Allow the 10x wash buffer to warm to room temperature before use until all salt crystals have dissolved. Calculate the amount of 1x wash buffer needed per assay. For each microplate, mix 50 mL of 10x wash buffer with 450 mL of distilled or deionized water. Mix thoroughly but gently.

[0789] 3. 1x mAb against fetuin-A with unique PTM (E103) Calculate the amount of 1x mAb against fetuin A with a unique PTM (E103) needed for each assay and mix the 1000x mAb against fetuin A with a unique PTM (E103) with the diluent according to the amount needed. For each microplate, mix 8 µL of 1000x mAb against fetuin A with a unique PTM (E103) with 8 mL of diluent. Mix evenly but gently.

[0790] 4.1x HRP conjugate Calculate the amount of 1X HRP conjugate needed for each assay and mix the 4000X HRP conjugate with the diluent accordingly. For each microplate, mix 3 µL of 4000X HRP conjugate with the 12 diluent. Mix thoroughly but gently.

[0791] Materials required but not provided Precision single micropipettes (1-10μL, 20-100μL, 100-200μL, 200-1000μL) and multichannel pipettes (100μL) Microcentrifuge tubes and disposable tips 500mL measuring cylinder Vortex mixer and microcentrifuge Orbital shaker Plastic containers for preparing reagents ·Microplate reader capable of measuring evaluation items at 450±10nm Distilled or deionized water Adhesive plate seal Reagent reservoir

[0792] Warnings and Precautions This test is for professional in vitro diagnostic use only. Do not use reagents that have passed their expiration date. Exposure to improper temperatures during all storage and assay procedures may adversely affect results. Do not reuse microplate wells. · Wear protective gloves during all assay procedures. Being light sensitive and a skin irritant, TMB substrate should be protected from direct light exposure and skin contact during all storage and assay procedures. Avoid skin contact with the stop solution containing 0.5N sulfuric acid, which may cause skin irritation and burns. · Consider all clinical specimens to be potentially infectious. Disposal of any waste materials should comply with local requirements and existing regulations regarding good laboratory practice.

[0793] Specimen collection and handling Morning urine samples should be collected in a clean, dry container to avoid cross-contamination. No additives or preservatives are required for urine sample integrity. Store urine samples at -20°C until use. Avoid repeated freezing and thawing of urine samples. Before performing the assay, allow the urine sample to come to room temperature. Centrifuge the urine sample at 1,000 ± 20 × g for 5 minutes. Remove the supernatant and test immediately. If necessary, use a diluent to dilute the sample.

[0794] Assay procedure Prepare enough microplate modules for all calibrators and urine samples and secure the microplates in the holder. Allow all reagents to come to room temperature before use.

[0795] 1. Mix calibrators 1-8 and each centrifuged urine sample with 1x mAb to E103 in a 1:1 ratio in a microcentrifuge tube (for triplicate tests, it is recommended to mix 180 μL of each calibrator / sample with 180 μL of 1x mAb to E103). Incubate on an orbital shaker at 25°C and 200 rpm for 2 hours.

[0796] 2. Transfer 100 μL of each incubated mixture into the designated well of an E103-coated microplate and incubate for 1.5 hours at 25° C. and 200 rpm on an orbital shaker. Keep the microplate covered and level during the entire incubation.

[0797] 3. After incubation, discard the contents of the wells.

[0798] 4. Wash each well with 300-400 μL of 1x Wash Buffer. Discard the contents and tap the well vigorously on absorbent paper to remove any residual liquid. Wash a total of four times.

[0799] 5. Add 100 μL of 1x HRP conjugate to each well. Incubate the microplate on an orbital shaker at 25°C and 200 rpm for 60 minutes.

[0800] 6. After incubation, discard the contents of the wells and wash the wells as described in step 4.

[0801] 7. Add 100 μL of TMB substrate to each well. Incubate at room temperature for 30 minutes in the dark.

[0802] 8. Add 100 μL of Stop Solution to each well. Mix by shaking briefly until the mixture is homogenous.

[0803] 9. Determine the absorbance at 450±10 nm within 30 minutes and calculate the results.

[0804] Calculating the results A 1.5-parameter or 4-parameter logistic curve fit is used to establish a calibration curve. The concentration of fetuin A with a unique PTM in a patient's urine sample can be calculated from the calibration curve by interpolation. The range of this assay is 7.813-500 ng / mL.

[0805] 2. Calculate the amount of fetuin-A with the unique PTM using urinary creatinine correction (ng / mg Cr).

[0806] Performance characteristics accuracy The precision of the Human uPTM3-DKD ELISA was evaluated in a 5-day study. Three human urine samples from known diabetic patients and five spiked human urine samples were used; samples were tested in triplicate, twice daily. Within-run precision: The coefficient of variation (CV) was calculated for each of the eight samples from the triplicate measurements in each run. Within-run precision: The coefficient of variation (CV) was calculated for each of the eight samples from the triplicate measurements in 10 separate runs.

[0807] The accuracy results are summarized in the table below. [Table 28]

[0808] Measurement range 7.813~500ng / mL

[0809] linearity The test was evaluated and the R 2 =0.99) and was found to be linear.

[0810] Detection limit Limit of blank (LoB) = 0.873 ng / mL Limit of detection (LoD) = 4.510 ng / mL Limit of quantification (LoQ)=18.983ng / mL [Table 29]

[0811] For example, Figure 27 shows a calibration curve for the Human uPTM3-DKD ELISA according to a non-limiting embodiment (calibration range 7.813 to 500 ng / mL).

[0812] Consideration In this cohort of 632 stable ...

Claims

1. 1. An assay kit for determining the likelihood of progressive renal function decline, comprising: a first solution comprising a first reagent for interacting with post-translationally modified fetuin-A fragments in urine fragments to indicate the level of said fetuin-A fragments in a urine sample from said kidney transplant recipient after kidney transplantation; a second solution comprising a second reagent for interacting with urinary creatinine to indicate the level of urinary creatinine in the urine sample; a device for determining the level of the fetuin-A fragment and the level of the urinary creatinine in the urine sample to determine a ratio of the determined level of the fetuin-A fragment to the determined level of the urinary creatinine, a ratio greater than about 20 ng / mg indicates a higher likelihood of graft failure over a period of about 10 years or less; the device, wherein the first solution and the second solution are the same solution or different solutions from each other; The assay kit comprising:

2. 2. The assay kit of claim 1, wherein said ratio greater than about 20 ng / mg indicates a need for medical intervention to forestall said graft failure.

3. 2. The assay kit of claim 1, wherein the graft failure is indicated by a doubling of serum creatinine, the need for kidney retransplantation, or the need for dialysis.

4. 2. The assay kit of claim 1, wherein the graft function decline is indicated by a decline in estimated glomerular filtration rate (eGFR).

5. 2. The assay kit of claim 1, wherein said interacting with said fetuin A fragment comprises binding to said fetuin A fragment.

6. 10. The assay kit of claim 1, wherein said interacting with said urinary creatinine comprises binding to said urinary creatinine.

7. 10. The assay kit of claim 1, wherein at least one of the first reagent and the second reagent comprises an antibody.

8. 2. The assay kit of claim 1, wherein the ratio greater than about 20 ng / mg is greater than about 25 ng / mg.

9. 2. The assay kit of claim 1, wherein the ratio greater than about 20 ng / mg is greater than about 30 ng / mg.

10. 2. The assay kit of claim 1, wherein the ratio greater than about 20 ng / mg is greater than about 35 ng / mg.

11. 2. The assay kit of claim 1, wherein the ratio greater than about 20 ng / mg is greater than about 40 ng / mg.

12. 2. The assay kit of claim 1, wherein the ratio greater than about 20 ng / mg is greater than about 50 ng / mg.

13. 2. The assay kit of claim 1, wherein the ratio greater than about 20 ng / mg is greater than about 70 ng / mg.

14. 2. The assay kit of claim 1, wherein the ratio greater than about 20 ng / mg is greater than about 90 ng / mg.

15. 2. The assay kit of claim 1, wherein the higher likelihood is greater than the likelihood of graft function decline over a period of about 10 years or less in a comparison recipient of a kidney transplant having a ratio of the determined level of fetuin A fragment to the level of urinary creatinine that is less than about 14 ng / mg.

16. 16. The assay kit of claim 15, wherein the second ratio less than about 14 ng / mg is less than about 13 ng / mg.

17. 16. The assay kit of claim 15, wherein the second ratio less than about 14 ng / mg is less than about 9 ng / mg.

18. 16. The assay kit of claim 15, wherein the second ratio less than about 14 ng / mg is less than about 7.5 ng / mg.

19. 16. The assay kit of claim 15, wherein the second ratio less than about 14 ng / mg is less than about 7 ng / mg.

20. 16. The assay kit of claim 15, wherein the second ratio less than about 14 ng / mg is less than about 4 ng / mg.

21. 2. The assay kit of claim 1, wherein the higher likelihood of graft failure is about 5% or greater.

22. 2. The assay kit of claim 1, wherein the higher likelihood of graft failure is about 10% or greater.

23. 2. The assay kit of claim 1, wherein the higher likelihood of graft failure is about 30% or greater.

24. The assay kit of claim 1 , wherein the graft failure occurs after two years.

25. The assay kit of claim 1 , wherein the graft failure occurs after 5 years.

26. The assay kit of claim 1, wherein the device is for determining the level of the fetuin A fragment and the level of the urinary creatinine to assess the probability of graft failure based on the ratio and at least one complementary marker.

27. 27. The assay kit of claim 26, wherein the at least one supplemental marker comprises age, sex, time since transplant at enrollment, urinary albumin to creatine ratio (UACR), estimated glomerular filtration rate (eGFR) based on the creatinine and cystatin C-based CKD-EPI equation, presence of human leukocyte antigen, or any combination thereof.

28. 28. The assay kit of claim 27, wherein the assessed probability exhibits an area under a receiver operating characteristic (ROC) curve (AUC) of at least about 70 percent (%).

29. 29. The assay kit of claim 28, wherein the assessed probability represents at least about 90 percent (%) of the AUC.

30. 29. The assay kit of claim 28, wherein the assessed probability represents at least about 95 percent (%) of the AUC.

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